Composite hydrogels for delivery of biologically active materials

ABSTRACT

A composite hydrogel comprises an amphiphilic triblock copolymer (ABA) and a loaded micelle bound by noncovalent interactions. The loaded micelle comprises a biologically active substance and an amphiphilic diblock copolymer (CD). The A blocks comprise a steroidal repeat unit (repeat unit 1) having both a backbone carbonate and a side chain bearing a steroid functional group. Each of the A blocks has a degree of polymerization of about 0.5 to about 4.0. The B block comprises a first poly(alkylene oxide) backbone. The C block comprises a second poly(alkylene oxide) backbone. The D block comprises a steroidal repeat unit (repeat unit 2) having both a backbone carbonate group and a side chain comprising a steroid functional group. The composite hydrogel is capable of controlled release of the biologically active substance.

PARTIES TO A JOINT RESEARCH AGREEMENT

This invention was made under a joint research agreement betweenInternational Business Machines Corporation and the Agency For Science,Technology and Research.

BACKGROUND

The present invention relates to composite hydrogels for delivery ofbiologically active materials, and more specifically to compositionscomprising hydrogel forming triblock copolymers and drug loaded micellesof diblock copolymers.

One of the major problems in the development of anticancer drugformulations is the delivery of the drugs with adequately highbioavailability for therapeutic intention. As many anticancer agents arehydrophobic, clinical administration of these drugs typically requiresdissolution using organic solvents. One such agent is paclitaxel, whichis a widely used small molecular drug effective against an extensiverange of solid tumors. However, its clinical applications have beenmostly impeded by its extremely low aqueous solubility (0.3micrograms/mL in water), which limits its administration to the use of aformulation comprising of 50:50 mixture of Cremophor EL (polyethoxylatedcastor oil):ethanol. Although this formulation is able to increase thesolubility and bioavailability of paclitaxel (PTX), it can also lead tohypersensitivity reactions and other severe side effects in some cases.Despite premedication with corticosteroids to reduce the immuneresponse, minor reactions such as rashes and flushing still occur in 41%to 44% of patients and potentially fatal reactions occur in 1.5% to 3%of patients. Thus, additional materials and methods are needed foradministering hydrophobic drugs.

Hydrogels have emerged as an important class of materials for biomedicalapplications due to their unique properties that bridge the gap betweensolid and liquid states. Amongst the numerous classes of syntheticpolymeric materials that are capable of forming hydrogels, ABA-typeamphiphilic triblock copolymers have a hydrophilic central B blockflanged on both the ends by peripheral hydrophobic A blocks. Thesematerials are of interest because they can potentially form physicalgels that do not involve the formation of covalent bonds. In someinstances, the self-assembled morphology can change from independentmicelles to a network of bridged micelles, and eventually to a hydrogelnetwork at higher polymer concentration.

Owing to their non-toxicity and biocompatibility, poly(ethylene glycol)(PEG) has been used extensively as the non-ionic hydrophilic B block inhydrogel forming ABA triblock copolymers. Homo- or copolymers ofpoly(lactides) and poly(caprolactone) have been used for the hydrophobicA blocks. A drawback of the reported triblock copolymers is the highconcentration and hydrophobic content needed for hydrogel formation. Forexample, gelation of an aqueous mixture ofpoly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide)(PLLA-b-PEG-b-PLLA) triblock copolymer occurs at a polymer concentrationof about 16 wt % (weight percent) based on total weight of the aqueousmixture. The hydrophobic block content of the triblock copolymer was ina range of about 17 wt % to 37 wt % based on total weight of thetriblock copolymer.

For many biomedical applications, it would be desirable to havepolymeric materials that can form hydrogels at a lower concentration,which can be utilized for the delivery of biologically active substancesthat include hydrophobic drugs.

SUMMARY

Accordingly, a composite hydrogel is disclosed, comprising:

an amphiphilic triblock copolymer (ABA), wherein i) the triblockcopolymer comprises two independent peripheral hydrophobic A blocks anda hydrophilic central B block, ii) each of the A blocks comprises asteroidal repeat unit (repeat unit 1) comprising both a backbonecarbonate and a side chain comprising a steroid functional group, iii)each of the A blocks has a degree of polymerization of about 0.5 toabout 4.0, and iv) the B block comprises a first poly(alkylene oxide)backbone; and

a loaded micelle comprising a biologically active substance and anamphiphilic diblock copolymer (CD), wherein i) the diblock copolymercomprises a hydrophilic C block and a hydrophobic D block, ii) the Cblock comprises a second poly(alkylene oxide) backbone, and iii) the Dblock comprises a steroidal repeat unit (repeat unit 2) comprising botha backbone carbonate group and a side chain comprising a steroidfunctional group;

wherein the amphiphilic triblock copolymer and the loaded micelle arebound by noncovalent interactions, and the composite hydrogel is capableof controlled release of a biologically active substance.

Also disclosed is a method, comprising:

mixing an amphiphilic triblock copolymer (ABA) with a loaded micelle,the loaded micelle comprising i) a biologically active substance and ii)an amphiphilic diblock copolymer (CD), thereby forming a compositehydrogel capable of controlled release of the biologically activesubstance;

wherein

the triblock copolymer (ABA) comprises two independent peripheralhydrophobic A blocks and a hydrophilic central B block, wherein i) eachof the A blocks comprises a steroidal repeat unit (repeat unit 1)comprising both a backbone carbonate and a side chain comprising asteroid functional group, ii) each of the A blocks has a degree ofpolymerization of about 0.5 to about 4.0, and iii) the B block comprisesa first poly(alkylene oxide) backbone,

the diblock copolymer (CD) comprises a hydrophilic C block and ahydrophobic D block, wherein i) the C block comprises a secondpoly(alkylene oxide) backbone, and ii) the D block comprises a steroidalrepeat unit (repeat unit 2) comprising both a backbone carbonate and aside chain steroid functional group, and

the triblock copolymer and the loaded micelle are bound by noncovalentinteractions.

Further disclosed is a composite hydrogel for controlled release of abiologically active substance, comprising:

an amphiphilic triblock copolymer (ABA) having the structure:

wherein each m is an independent number having a value of about 0.8 toabout 2.5, and n′ has a value of about 200 to about 800; and

a loaded micelle comprising i) the biologically active substance and ii)an amphiphilic diblock copolymer (CD) having the structure:

wherein n has a value of about 100 to about 120, x has a value of about11, and y has a value of about 30, and wherein the amphiphilic triblockcopolymer and the loaded micelle are bound by noncovalent interactions

Also disclosed is a triblock copolymer having the formula (19)

wherein

each m is an independent number having a value of about 0.8 to about 4,

n′ has a value of about 200 to about 800,

each t is an independent integer having a value of 0 to 6,

each t′ is an independent integer having a value of 0 to 6,

t′ and t cannot both be zero in any repeat unit,

each Q¹ is an independent monovalent radical selected from the groupconsisting of hydrogen, halides, alkyl groups comprising 1 to 30carbons, and aryl groups comprising 6 to 30 carbon atoms,

each L″ is an independent linking group selected from the groupconsisting of a single bond and divalent radicals comprising 1 to 30carbons, and

each S′ is an independent steroid group.

The above-described and other features and advantages of the presentinvention will be appreciated and understood by those skilled in the artfrom the following detailed description, drawings, and appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a graph showing the intensity ratio (1339/1334) versuslogarithm of concentration (mg/L) of block copolymers (Examples 4, and 6to 9), respectively, in deionized (DI) water. These data were used todetermine critical micelle concentrations (CMC) values of Examples 4,and 6 to 9 of 2.1 mg/L, 2.1 mg/L, 1.5 mg/L, 1.5 mg/L and 2.1 mg/L,respectively (Table 8).

FIGS. 2A to 2F are transmission electron micrograph (TEM) images ofaqueous micelles formed by the block copolymers of Examples 4 to 9,respectively. The upper image in FIG. 2F is at a magnification of28000×. The lower image in FIG. 2F is at a magnification of 110000×.

FIG. 3 is a graph showing human liver carcinoma HepG2 cell viability (%)against block copolymer concentration (Examples 4, and 6 to 9). Theblock copolymers are non-cytotoxic. More than 90% cell viability wasobserved even at the highest polymer concentration (2377 mg/L).

FIGS. 4A to 4E are TEM images of paclitaxel loaded micelles (PTX-loadedmicelles) of Examples 11 to 15.

FIG. 5A is a graph showing the cumulative release of PTX from thePTX-loaded micelles (Examples 11 to 15, Table 9) with time.Approximately 100% of the PTX was released within 24 hours from loadedmicelles PTX-Polymer(11:0) (Example 12). PTX release was more sustainedwith the remaining loaded micelles (Example 11, and Examples 13 to 15),displaying 70% to 95% release by 144 hours.

FIG. 5B is a graph showing the cumulative release of the drugcyclosporin A (CYC) from the CYC-loaded micelles (Examples 16 to 18,Table 10) with time. CYC release was sustained from micellar formulationCYC-Polymer(11:30), Example 18, for about 5 days.

FIG. 5C is a graph showing the cumulative release of the drugspironolactone (SPL) from the SPL-loaded micelles (Examples 19 to 21,Table 11) with time. SPL release from micellar formulationSPL-Polymer(11:30), Example 21, was sustained for about 7 hours.

FIG. 6 is a bar chart comparing the viability of HepG2 cells afterincubation with PTX-loaded micelles (Examples 11 to 15) and PTX alonefor 48 hours at various PTX concentrations. The loaded micellePTX-Polymer(8:8) (Example 13) killed HepG2 cells more efficiently thanthe free PTX at most PTX concentrations. A similar trend was observedwith loaded micelle PTX-Polymer(11:0) (Example 12) at low PTXconcentrations. The killing efficiency of loaded micellesPTX-Polymer(0:67) (Example 11), PTX-Polymer(11:30) (Example 14), andPTX-Polymer(18:55) (Example 15) against HepG2 cells was greater thanfree PTX at all concentrations tested.

FIG. 7 is a graph of intensity ratio (1337/1334) vs. logarithm ofconcentration (mg/L) of TRI(35:2.4) in DI water.

FIG. 8 is a graph of the frequency sweep of storage (G′) and loss (G″)moduli at 25° C. of triblock copolymer TRI(20:0.8) at 5 wt % and 8 wt %concentration in DI water, and composite hydrogel formed withTRI(20:2.2) at 5 wt % concentration.

FIG. 9 is a graph of the frequency sweep of storage (G′) and loss (G″)moduli at 25° C. of triblock copolymer TRI(35:2.4) at 4 wt %concentration in DI water, and composite hydrogel formed withTRI(35:2.4) at 4 wt % concentration

FIG. 10 is a graph of the frequency sweep of storage (G′) and loss (G″)moduli at 37° C. of triblock copolymer TRI(35:2.4) at 4 wt %concentration in DI water, and composite hydrogel formed withTRI(35:2.4) at 4 wt % concentration.

FIG. 11 is a graph of the flow sweep of 4 wt % TRI(35:2.4) at 25° C. and37° C., demonstrating the shear thinning behavior of the hydrogel.

FIG. 12 is an SEM image of cryo-fixed TRI(35:2.4) (4 wt %) hydrogel. Thesample was prepared by swelling the polymer with DI water untilequilibrium and rapidly transferring into a chamber filled with liquidnitrogen. This was then followed by a 2-day freeze-drying process.

FIG. 13 is an SEM image of cryo-fixed TRI(35:2.4) (4 wt %) hydrogel. Thesample was prepared by swelling the Polymer(11:30) micelle suspensionnot loaded with drug until equilibrium and rapidly transferred into achamber filled with liquid nitrogen. This was then followed by a 2-dayfreeze-drying process.

FIG. 14 is a photograph of a composite hydrogel containing fluoresceinisothiocyanate (FITC) (left) (Example 30) and free FITC in hydrogel(right) (Example 33). The left side is lighter because of the greenfluorescence of FITC. The right side is darker because of the absence ofFITC fluorescence. It can be seen that the composite gel containingFITC-loaded micelles (Example 30) was able to fluoresce underUV-illumination at 365 nm, while no fluorescence could be seen from freeFITC-containing hydrogel (Example 33). This indicates the difference insolvent microenvironment for FITC which results in changes to itsfluorescence characteristics.

FIG. 15 is a graph showing FITC release as a function of time fromvarious samples: composite hydrogel containing FITC (Example 30), freeFITC in hydrogel (Example 33), and FITC-loaded micelles alone (Example27).

DETAILED DESCRIPTION

Composite hydrogels are disclosed that comprise an amphiphilic ABAtriblock copolymer (ABA copolymer) and a loaded micelle, which are boundby non-covalent interactions. The triblock copolymer comprises ahydrophilic central B block, and two independent hydrophobic peripheralA blocks. The A blocks have a steroidal repeat unit comprising a steroidfunctionalized side chain and a backbone carbonate group. The central Bblock has a poly(alkylene oxide) backbone. The loaded micelle comprisesan amphiphilic diblock copolymer (referred to as CD copolymer) and abiologically active substance, also bound by non-covalent interactions.The CD copolymer has a hydrophilic C block and a hydrophobic D block.The C block has a poly(alkylene oxide) backbone and the D block has asteroidal repeat unit comprising a steroid functionalized side chain anda carbonate backbone group. The steroidal repeat units of the A blockand D block can be the same or different steroidal repeat units. Todistinguish these steroidal repeat units in the following description,the steroidal repeat unit of the A block is referred to as repeat unit 1and the steroidal repeat unit of the D block is referred to as therepeat unit 2. The A block and/or the D block can include additionalrepeat units having a carbonate and/or ester backbone functional groups.The examples further below demonstrate that the composite hydrogelscomprising a micelle loaded with fluorescein isothiocyanate (FITC),which is used as a model for a drug, can release the FITC over a longerperiod compared to the loaded micelles alone. Thus, the compositehydrogels provide a straightforward and modular approach for theencapsulation and controlled release of biologically active materialsfor a variety of applications including but not limited to hydrophobicdrug delivery, tissue engineering, and/or the design of cosmetics/foodproducts additives. For instance, the composite hydrogels can be used toencapsulate molecules that can serve as active ingredients forcosmetic/dietary applications. These molecules can be small moleculessuch as vitamins or large biomolecules such as proteins. The ABAcopolymer and CD copolymer are biodegradable and/or biocompatible.

The ABA copolymer has a low hydrophobic content, meaning repeat unit 1of the A block is present in an amount of about 5 wt % to about 15 wt %based on total weight of the ABA copolymer. The ABA copolymer is notnecessarily capable of forming a hydrogel alone in water. In someinstances, the ABA copolymer is insoluble in water, but will form ahydrogel upon mixing with the loaded micelle. In instances when the ABAcopolymer alone can form a hydrogel in water, the ABA hydrogel can format an ABA copolymer concentration of about 4 wt %. The storage modulus(G′) of the ABA hydrogel can be tuned from about 300 Pa to about 3500 Paby varying the ABA copolymer composition and/or concentration.

The composite hydrogels can also form at a total solids content of about4 wt % to about 8 wt % based on total weight of the aqueous mixture.

The ABA copolymer and CD copolymer are preferably derived from cycliccarbonate monomers comprising pendant steroid groups, referred to hereinas steroidal monomers, which undergo organocatalyzed ring-openingpolymerization (ROP). The resulting copolymers are functionalized with aside chain steroid group. The ring opening polymerization is initiatedwith a polymeric initiator comprising i) one or two nucleophilicinitiator groups selected from the group consisting of alcohols, amines,thiols and combinations thereof, and ii) a poly(alkylene oxide)backbone.

For a given initiator, the amphiphilic properties of the blockcopolymers can be controlled by adjusting the amount and structure ofthe various repeat units in the ring opened polymer chain, that is, byadjusting the amount and structure of the cyclic carbonate monomerbearing a steroid group and/or the amount and structure of any diluentcyclic carbonyl monomer (cyclic carbonate and/or cyclic estermonomer(s)) used in the ring opening polymerization. The diluent cycliccarbonyl monomer does not comprise a steroid group. Thus, thehydrophobic A blocks and the hydrophobic D block can comprise apolycarbonate and/or polyestercarbonate chain bearing a side chainsteroid group.

The ABA copolymer has a low critical micelle concentrations (CMC) inwater. The CMC can have a value of 1.0 mg/L to 50 mg/L, 1.0 mg/L to 10mg/L, and more particularly 1.0 mg/L to 2.5 mg/L.

The CD copolymer also has a low critical micelle concentrations (CMC) inwater. The CMC can have a value of 1.0 mg/L to 50 mg/L, 1.0 mg/L to 10mg/L, and more particularly 1.0 mg/L to 2.5 mg/L.

The CD copolymer is capable of forming reversible nanoparticulatecomplexes with hydrophobic biologically active substances (e.g., drugsand/or genes) by noncovalent interactions. For example, the CD copolymercan encapsulate the rigid hydrophobic anticancer drug paclitaxel (PTX)in the form of a water dispersible nano-sized particle (i.e., loadedmicelle). Paclitaxel has the structure:

The loaded micelle forms without sonication or homogenization and has anarrow particle size distribution. The loaded micelle comprises PTX inan amount more than 0 wt %, and more particularly in an amount of 0.1 wt% to 15 wt % based on total dry weight of the loaded micelle. Otherhydrophobic drugs that can be encapsulated by the amphiphilic blockcopolymers include cyclosporin A (CYC, an immunosuppressive agent usedafter organ transplantation) and spironolactone (SPL, a drug used totreat hypertension). The drugs can be used singularly or in combination.CYC and SPL have the structures (stereochemistry shown):

In the following description, the term “cyclic carbonyl monomer”includes cyclic carbonate monomers, cyclic ester monomers, andcombinations thereof that can be polymerized in a ring openingpolymerization.

Herein, a cyclic carbonate monomer having a pendant steroid group isreferred to as a “steroidal monomer.” A second cyclic carbonyl monomer,referred to as a “diluent monomer,” does not contain a steroid group.The diluent monomer can be a cyclic carbonate monomer, a cyclic ester(i.e., lactone, lactide) monomer, or a combination thereof. Thesteroidal monomer can be used singularly or in combination with one ormore other steroidal monomers. The diluent monomer can be usedsingularly or in combination with one or more other diluent monomers.

A repeat unit having a pendant steroid group is referred to generally asa “steroidal repeat unit.” Herein, a steroidal repeat unit has acarbonate backbone group. A repeat unit that does not contain a steroidgroup is referred to as a “diluent repeat unit.” The diluent repeat unitcan have a carbonate and/or ester backbone group.

In an embodiment, each of the two A blocks of the ABA copolymer consistsessentially of repeat unit 1. The A blocks can independently have adegree of polymerization of about 0.5 to 10, more specifically 0.5 to 4,even more specifically 0.8 to 2.4 of repeat unit 1. It should beunderstood that the degree of polymerization represents an average valuebased on the proton nuclear magnetic resonance (¹H NMR) spectroscopyand/or SEC of the ABA copolymer.

Preferably, the hydrophobic D block of the CD copolymer comprises, inaddition to repeat unit 1 having a side chain steroid group, a diluentrepeat unit comprising a carbonate or ester backbone group.

In an embodiment, the ABA copolymer and CD copolymer comprise the samesteroidal pendant group. In another embodiment, repeat unit 1 of the ABAcopolymer is the same as repeat unit 2 of the CD copolymer.

The term “biodegradable” is defined by the American Society for Testingand Materials as degradation caused by biological activity, especiallyby enzymatic action, leading to a significant change in the chemicalstructure of the material. For purposes herein, a material is“biodegradable” if it undergoes 60% biodegradation within 180 days inaccordance with ASTM D6400. Herein, a material is “enzymaticallybiodegradable” if the material can be degraded (e.g., depolymerized) bya reaction catalyzed by an enzyme.

A “biocompatible” material is defined herein as a material capable ofperforming with an appropriate host response in a specific application.

The steroidal monomer and the diluent monomer can be stereospecific ornon-stereospecific. A stereospecific monomer or a stereospecific repeatunit i) has a non-superposable mirror image and ii) comprises one ormore asymmetric tetravalent carbons (i.e., tetrahedral sp³ carbons).Each asymmetric tetravalent carbon is assigned an R or S symmetry basedon Cahn-Ingold-Prelog (CIP) symmetry rules. For example, if astereospecific repeat unit has one asymmetric tetravalent carbon, thenthe stereospecific repeat unit can be present substantially as the Rstereoisomer or substantially as the S stereoisomer, meaning thestereoisomer can be present in a stereoisomeric purity of 90% to 100%,94% or more, or more particularly 98% to 100%. In another example, ifthe stereospecific repeat unit has two asymmetric tetravalent carbons,the stereospecific repeat unit can be present substantially as the R,Rstereoisomer, substantially as the R,S stereoisomer, substantially asthe S,S stereoisomer, or substantially as the S,R stereoisomer.

A stereospecific cyclic carbonyl monomer i) has a non-superposablemirror image and ii) comprises one or more asymmetric tetravalentcarbons. The stereospecific cyclic carbonyl monomer has a stereoisomericpurity of 90% or more, and more particularly 98% or more. The asymmetrictetravalent carbons of the stereospecific cyclic carbonyl monomer can bein the steroid group or in a ring carbon that becomes a polymer backbonecarbon in the ring opening polymerization.

“Restricted metals” herein include ionic and nonionic forms ofberyllium, magnesium, calcium, strontium, barium, radium, aluminum,gallium, indium, thallium, germanium, tin, lead, arsenic, antimony,bismuth, tellurium, polonium, and metals of Groups 3 to 12 of thePeriodic Table. Metals of Groups 3 to 12 of the Periodic Table includescandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium,ruthenium, rhodium, palladium, silver, cadmium, lanthanum, cerium,praseodymium, neodymium, promethium, samarium, europium, gadolinium,terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium,hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold,mercury, actinium, thorium, protactinium, uranium, neptunium, plutonium,americium, curium, berkelium, californium, einsteinium, fermium,mendelevium, nobelium, lawrencium, rutherfordium, dubnium, seaborgium,bohrium, hassium, meitnerium, darmstadtium, roentgenium, andcopernicium. Each one of the foregoing restricted metals can have aconcentration in the block copolymer of 0 parts to 100 ppm (parts permillion), 0 parts to 100 ppb (parts per billion), or 0 parts to 100 ppt(parts per trillion). Preferably, each one of the foregoing restrictedmetals has a concentration of 0 parts in the ABA and CD copolymers(i.e., the concentration is below detection limits). In an embodiment,the chemical formulas of the components used in the ring openingpolymerization, including the steroidal monomer, the diluent monomer,the polymeric initiator, the catalyst for the ring openingpolymerization, the solvent, and any base accelerator, contain none ofthe above restricted metals. The biologically active cargo material cancomprise a restricted metal.

No restriction is placed on the concentration of boron, silicon, or anyindividual alkali metal in the ABA and CD copolymers.

The term “carrier” as used herein refers to the combination of the ABAand CD copolymers. The carrier can be biologically active when usedalone (e.g., as a result of enzymatic release of the covalently boundsteroid group, where the released steroid performs a therapeuticfunction). The composite hydrogel can therefore serve one or morebiological functions, including providing enhanced biological activityof either the released steroid and/or the released biologically activesubstance (i.e., cargo material). As an example, upon contacting atissue (e.g., wound surface) or cell, the composite hydrogel can releasethe biologically active substance and/or the steroid at separate ratesand times over a desired period. Alternatively or additionally, thecomposite hydrogel can enter a tissue or a cell (e.g., by endocytosis),and at a desired stage release the biologically active substance and/orthe steroid within the tissue or the cell.

The biologically active substance can be any suitable biologicallyactive substance that complexes with the CD copolymer in water bynon-covalent interactions to form a loaded micelle. Biologically activesubstances include cells, biomolecules (e.g., DNA, genes, peptides,proteins, enzymes, lipids, phospholipids, and nucleotides), natural orsynthetic organic compounds (e.g., drugs, dyes, synthetic polymers,oligomers, and amino acids), inorganic materials (e.g., metals and metaloxides), radioactive variants of the foregoing, and combinations of theforegoing. In an embodiment, the biologically active substance is a drugand/or a gene.

“Biologically active” means the referenced material can alter thechemical structure and/or activity of a cell in a desirable manner, orcan selectively alter the chemical structure and/or activity of a celltype relative to another cell type in a desirable manner. As an example,one desirable change in a chemical structure can be the incorporation ofa gene into the DNA of the cell. A desirable change in activity can bethe expression of the transfected gene. Another change in cell activitycan be the induced production of a desired hormone or enzyme.Alternatively, a desirable change in activity can be the selective deathof one cell type over another cell type. No limitation is placed on therelative change in cellular activity caused by the biologically activesubstance, providing the change is desirable and useful. Moreover, nolimitation is placed on the cargo, providing the cargo induces a usefulcellular response when released from the composite hydrogel.

The steroidal monomer is a cyclic carbonate monomer comprising a pendantsteroid group. The steroid group can be a chemical moiety derived from anaturally occurring human steroid, non-human steroid, and/or syntheticsteroid compound. Herein, a steroid group comprises a tetracyclic ringstructure according to formula (1):

wherein the 17 carbons of the ring system are numbered as shown. Eachring of the tetracyclic ring structure can independently comprise one ormore double bonds. The steroid group can comprise one or moresubstituent groups independently comprising 0 to 30 carbons attached toone or more of the numbered ring positions of the tetracyclic ringstructure.

The steroidal monomer comprises a cyclic carbonate ring that is joinedto a steroid group by a divalent linking group L′. The steroidal monomerhas the formula (2):

wherein t and t′ are independent integers having a value of 0 to 6,wherein t′ and t cannot both be zero, and each Q¹ is a monovalentradical independently selected from the group consisting of hydrogen,halides, alkyl groups comprising 1 to 30 carbons, and aryl groupscomprising 6 to 30 carbon atoms. Ring carbons and oxygens are numberedas shown in formula (2). L′ is a divalent linking group comprising oneor more carbons. S′ is a steroid group. The L′-S′ bond is preferablyhydrolytically cleavable and/or enzymatically cleavable, meaning thebond can be directly or indirectly cleaved as a result of enzymaticactivity. Each Q¹ group can independently be branched or non-branched.Each Q¹ group can also independently comprise one or more additionalfunctional groups selected from the group consisting of ketones,aldehydes, alkenes, alkynes, cycloaliphatic rings comprising 3 to 10carbons, heterocylic rings comprising 2 to 10 carbons, ethers, amides,esters, and combinations of the foregoing functional groups. Aheterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two ormore Q¹ groups can together form a ring. The steroidal monomer can bestereospecific or non-stereospecific. In an embodiment, t and t′ areeach 1, each Q¹ at carbon 4 is hydrogen, each Q¹ at carbon 6 ishydrogen, and Q¹ at carbon 5 is selected from the group consisting ofhydrogen, methyl, and ethyl. In another embodiment, S′ is a cholesterylgroup.

L′ can comprise one or more functional groups selected from the groupconsisting of ketone, ester, amide, imide, thioester, carbamate,thiocarbamate (i.e., —S—C═O(N(R)—), urea, anhydride, and combinationsthereof. In an embodiment, L′ comprises a carbonyl group linked to S′.In another embodiment, L′ comprises an amidocarbonyl group:

wherein R′ is hydrogen or a monovalent radical comprising 1 to 5carbons, and the carbonyl group of the amidocarbonyl group is linked toS′.

Exemplary steroid groups S′ include but are not limited tostereospecific structures such as:

from cholesterol and referred to herein as a cholesteryl group,

from testosterone,

from equilin,

from epiandrosterone,

from dihydrotestosterone,

from nandrolone,

from dihydroprogesterone,

from pregnenolone,

from equilenin,

from dehydroepiandrosterone,

from corticosterone acetate,

from deoxycorticosterone, and

from estrone.In the above steroid groups, the starred bond represents the attachmentpoint to L′. The R,S stereochemistry of each asymmetric tetravalentcarbon is indicated in the above steroid groups. Other steroid groupsinclude various stereoisomers of the foregoing steroid groups. Thesteroid groups can be used in combination, meaning two or more steroidalmonomers differing in chemical structure and/or in stereospecificity canbe used to form the polycarbonate block of the amphiphilic blockcopolymer.

Ring opening polymerization of the steroidal monomer of formula (2)produces a steroidal repeat unit having the formula (3):

wherein L′, t, t′, Q¹, and S′ are defined as above. Atom numbers of thebackbone carbons and oxygens are indicated in formula (3). The steroidalrepeat unit comprises a backbone carbonate group. The starred bonds informula (3) represent attachment points to other subunits or to aterminal functional group of the ROP polymer chain.

More specific steroidal monomers include cyclic carbonate compounds offormula (4):

wherein t, t′, Q¹, and S′ are defined as above. L″ can be a single bond(i.e., S′ is linked to the ester oxygen of formula (4) by a singlebond). Alternatively, L″ can be a divalent linking group comprising 1 to30 carbons. The L″-S′ bond can be a hydrolytically and/or enzymaticallycleavable bond.

Ring opening polymerization of the steroidal monomer of formula (4)produces a steroidal repeat unit having the formula (5):

wherein L″, t, t′, Q¹, and S′ are defined as above, and backbone carbonsand oxygens are numbered as indicated. The steroidal repeat unitcomprises a backbone carbonate group.

More specific steroidal monomers of formula (2) include cholesterylmonomers of formula (6):

wherein L′, t, t′ and Q¹ are defined as in formula (2). The R,Sstereochemistry of each asymmetric tetravalent carbon center of thecholesteryl moiety is indicated in formula (6). In an embodiment, theL′-0 bond in formula (6) is hydrolytically and/or enzymaticallycleavable.

The steroidal repeat unit formed by a ring opening polymerization of thecholesteryl monomer of formula (6) has the formula (7):

wherein L′, t, t′, and Q¹ are defined as in formula (2), and backbonecarbons and oxygens are numbered as shown. The R,S stereochemistry ofeach asymmetric tetravalent carbon center of the cholesteryl moiety isindicated in formula (7). The steroidal repeat unit of formula (7)comprises a backbone carbonate group.

More specific steroidal monomers of formula (4) include cholesterylmonomers of formula (8):

with R,S stereochemistry shown, wherein L″ is defined as above underformula (4), and t, t′, and Q¹ are defined as above under formula (2).

The steroidal repeat unit formed by ring opening polymerization of thecholesteryl monomer of formula (8) has the formula (9):

with R,S stereochemistry shown, wherein L″, t, t′, and Q¹ are defined asabove, and backbone carbons and oxygens are numbered as shown. Thesteroidal repeat unit of formula (9) comprises a backbone carbonategroup.

In an embodiment, L′ has a structure according to formula (10):

wherein L′″ is a divalent linking group comprising 1 to 20 carbons. L′″can comprise one or more heteroatoms selected from the group consistingof oxygen, nitrogen, sulfur, and combinations thereof. In an embodimentL′″ has the formula *—O—CH₂(CH₂)_(x)CH₂N(R)—*, wherein x is an integerof 1 to 10 and R′ is an monovalent alkyl group having 1 to 5 carbons:

The diluent monomer, which forms a diluent repeat unit of the ROPpolymer chain, can be selected from cyclic carbonate compounds of theformula (11):

wherein u and u′ are independent integers having a value of 0 to 6wherein u and u′ cannot both be zero, and each Q² is a monovalentradical independently selected from the group consisting of hydrogen,halides, alkyl groups comprising 1 to 30 carbons, and aryl groupscomprising 6 to 30 carbon atoms. Ring carbons and oxygens are numberedas shown in formula (11). No Q² group comprises a steroid group. Each Q²group can independently be branched or non-branched. Each Q² group canindependently be stereospecific or non-stereospecific. Each Q² group canindependently comprise one or more additional functional groups selectedfrom the group consisting of ketones, aldehydes, alkenes, alkynes,ethers, amides, esters, cycloaliphatic rings comprising 3 to 10 carbons,heterocylic rings comprising 2 to 10 carbons, and combinations of theforegoing functional groups. A heterocyclic ring can comprise oxygen,sulfur and/or nitrogen. Two or more Q² groups can together form a ring.In an embodiment, u′ and u are each 1, and each Q² is hydrogen (i.e.,trimethylene carbonate).

The diluent repeat unit formed by ring opening polymerization of thediluent monomer of formula (11) has the formula (12):

wherein u, u′, and Q² are defined as above under formula (11), andbackbone carbons and oxygens are numbered as shown. In this instance,the diluent repeat unit comprises a backbone carbonate group.

More specific diluent monomers of formula (11) have the formula (13):

wherein Q² is defined as above under formula (11), R² is a monovalentradical independently selected from the group consisting of alkyl groupscomprising 1 to 30 carbons and aryl groups comprising 6 to 30 carbons,and R² does not comprise a steroid group.

The diluent repeat unit formed by ring opening polymerization of thediluent monomer of formula (13) has the formula (14):

wherein Q² and R² are defined as above, and backbone carbons and oxygensare numbered as shown. This diluent repeat unit also comprises abackbone carbonate group.

The diluent monomer can be selected from cyclic ester monomers (e.g.,lactones). Exemplary cyclic ester monomers include compounds of theformula (15):

wherein v is an integer of 1 to 8, each Q³ is a monovalent radicalindependently selected from the group consisting of hydrogen, halides,alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to30 carbon atoms. No Q³ group comprises a steroid group. The cyclic esterring can optionally comprise a carbon-carbon double bond; that is,optionally, a

group of formula (15) can independently represent a

group. The cyclic ester ring can also comprise a heteroatom such asoxygen, nitrogen, sulfur, or a combination thereof; that is, optionallya

group of formula (15) can independently represent a *—O—*, *—S—*,*—N(H)—*, or an *—N(R¹)—* group, wherein R¹ is a monovalent radicalindependently selected from the group consisting of alkyl groupscomprising 1 to 30 carbons and aryl groups comprising 6 to 30 carbons.Cyclic ester monomers of formula (15) can be stereospecific ornon-stereospecific.

The diluent repeat unit formed by ring opening polymerization of thediluent monomer of formula (15) has the formula (16):

wherein Q³ and v are defined as above under formula (15). This diluentrepeat unit comprises a backbone ester group.

Herein, a ring opened polymer chain that has only backbone carbonategroups is a polycarbonate. A ring opened polymer chain that has backbonecarbonate groups and backbone ester groups is referred to as apolyestercarbonate.

The diluent monomer can be selected from a dioxane dicarbonyl monomersof the formula (17):

wherein w and w′ are independent integers having a value of 1 to 3, andeach Q⁴ and each Q⁵ is a monovalent radical independently selected fromthe group consisting of hydrogen, halides, alkyl groups comprising 1 to30 carbons, and aryl groups comprising 6 to 30 carbon atoms. No Q⁴ groupand no Q⁵ group comprises a steroid group. Compounds of formula (17) canbe stereospecific or non-stereospecific. In an embodiment, w and w′ areeach 1, each Q⁴ is hydrogen, and each Q⁵ is an alkyl group comprising 1to 6 carbons. In another embodiment, the diluent monomer is D-lactide orL-lactide.

The diluent repeat unit formed by ring opening polymerization of thediluent monomer of formula (17) has the formula (18):

wherein Q⁴, Q⁵, w, and w′ are defined as above under formula (17), andbackbone carbons and oxygens are numbered as shown.

Non-limiting examples of diluent monomers of formulas (11) and (13)include the cyclic carbonate monomers of Table 1.

TABLE 1

m = 1: Trimethylene carbonate (TMC) m = 2: Tetramethylene carbonate(TEMC) m = 3: Pentamethylene carbonate (PMC)

R = hydrogen (MTCOH) R = methyl (MTCOMe) R = t-butyl (MTCO^(t)Bu) R =ethyl (MTCOEt)

(MTCCl)

(MTCOBn)

(MTCTFE)

R = methyl R = iso-propyl

(MTCOEE)

(MTCOEtI)

(MTCOPrCl)

(MTCOPrBr)

MTCU

Non-limiting examples of diluent monomers having a cyclic esterstructure of formula (15) include the compounds of Table 2, andstereospecific versions thereof where feasible.

TABLE 2

R = H; n = 1: beta-Propiolactone (b-PL) R = H; n = 2:gamma-Butyrolactone (g-BL) R = H; n = 3: delta-Valerolactone (d-VL) R =H; n = 4: epsilon-Caprolactone (e-CL) R = CH₃; n = 1: beta-Butyrolactone(b-BL) R = CH₃; n = 2: gamma-Valerolactone (g-VL)

Pivalolactone (PVL)

1,5-Dioxepan-2-one (DXO)

5-(Benzyloxy)oxepan-2-one (BXO)

7-Oxooxepan-4-yl 2-bromo-2- methylpropanoate (BMP-XO)

5-Phenyloxepan-2-one (PXO)

5-Methyloxepan-2-one (MXO)

1,4,8-Trioxa(4,6)spiro-9-undecane (TOSUO)

5-(Benzyloxymethyl)oxepan-2-one (BOMXO)

7-Oxooxepan-4-yl 3-hydroxy-2- (hydroxymethyl)-2-methylpropanoate(OX-BHMP)

(Z)-6,7-Dihydrooxepin-2(3H)-one (DHXO)

Examples of diluent monomers of formula (17) include the compounds ofTable 3.

TABLE 3

D-Lactide (DLA), L-Lactide (LLA), or racemic Lactide, 1:1 D:L forms(DLLA)

meso-Lactide (MLA) (two opposite centers of asymmetry, R and S)

Glycolide (GLY)

The above monomers can be purified by recrystallization from a solventsuch as ethyl acetate or by other known methods of purification, withparticular attention being paid to removing as much water as possiblefrom the monomer. The monomer moisture content can have a value of 1 to10,000 ppm, 1 to 1,000 ppm, 1 to 500 ppm, and most specifically 1 to 100ppm, by weight of the monomer.

In an embodiment, the ABA triblock copolymer has a structure accordingto formula (19):

wherein

each m is an independent number having a value of about 0.8 to about 4,

n′ has a value of about 200 to about 800,

each t is an independent integer having a value of 0 to 6,

each t′ is an independent integer having a value of 0 to 6,

t′ and t cannot both be zero in any repeat unit,

each Q¹ is an independent monovalent radical selected from the groupconsisting of hydrogen, halides, alkyl groups comprising 1 to 30carbons, and aryl groups comprising 6 to 30 carbon atoms,

each L′ is an independent linking group selected from the groupconsisting of a single bond and divalent radicals comprising 1 to 30carbons, and

each S′ is an independent steroid group.

In another embodiment, the ABA triblock copolymer has a structureaccording to formula (20):

wherein

each m is an independent number having a value of about 0.8 to about 4,

n′ has a value of about 200 to about 800,

each t is an independent integer having a value of 0 to 6,

each t′ is an independent integer having a value of 0 to 6,

t′ and t cannot both be zero in any repeat unit,

each Q¹ is an independent monovalent radical selected from the groupconsisting of hydrogen, halides, alkyl groups comprising 1 to 30carbons, and aryl groups comprising 6 to 30 carbon atoms,

each L″ is an independent linking group selected from the groupconsisting of a single bond and divalent radicals comprising 1 to 30carbons, and

each S′ is an independent steroid group.

More specifically, the ABA triblock copolymer can have a structureaccording to formula (21):

wherein each m is an independent number having a value of about 0.8 toabout 4.0, and n′ has a value of about 200 to about 800.ROP Initiators

In general, initiators for ring opening polymerizations includenucleophilic groups such as alcohols, primary amines, secondary amines,and thiols. The ABA copolymer is preferably formed using adinucleophilic poly(alkylene glycol) initiator (e.g., poly(ethyleneglycol) (PEG)), and the CD copolymer is preferably formed using amono-endcapped mono-nucleophilic poly(alkylene glycol) initiator (e.g.,mono-methyl poly(ethylene glycol) (mPEG-OH)). The ABA copolymer derivedfrom PEG has a central B block comprising a poly(ethylene oxide) chainlinked at each end to an A block. The CD copolymer derived from mPEG-OHhas a C block comprising a mono-endcapped poly(ethylene oxide) chainlinked at one end to the D block.

The polymeric initiator can comprise a nucleophilic chain end groupindependently selected from the group consisting alcohols, primaryamines, secondary amines, and thiols, such as mono-endcapped PEG-diaminerepresented by the structure

wherein E″ represents a derivative of a terminal aminoethylene group,and monoendcapped PEG-dithiol, represented by the formula (22):

wherein E′″ represents a derivative of a terminal thioethylene group.

The number average molecular weight (Mn) of the mono-nucleophilicpolyether initiator can have a value of 100 to 100,000, more preferably100 to 10000, and even more preferably 100 to 5000.

Ring Opening Polymerizations (ROP)

The following description of methods, conditions and materials for ringopening polymerizations is applicable to the preparation of the blockcopolymer.

The ring-opening polymerization can be performed at a temperature thatis about ambient temperature or higher, more specifically 15° C. to 200°C., and even more specifically 20° C. to 80° C. When the reaction isconducted in bulk, the polymerization is performed at a temperature of50° C. or higher, and more particularly 100° C. to 200° C. Reactiontimes vary with solvent, temperature, agitation rate, pressure, andequipment, but in general the polymerizations are complete within 1 to100 hours.

The ROP reaction can be performed with or without the use of a solvent.Optional solvents include dichloromethane, chloroform, benzene, toluene,xylene, chlorobenzene, dichlorobenzene, benzotrifluoride, petroleumether, acetonitrile, pentane, hexane, heptane, 2,2,4-trimethylpentane,cyclohexane, diethyl ether, t-butyl methyl ether, diisopropyl ether,dioxane, tetrahydrofuran, or a combination comprising one of theforegoing solvents. When a solvent is present, a suitable monomerconcentration is about 0.1 to 5 moles per liter, and more particularlyabout 0.2 to 4 moles per liter.

Whether performed in solution or in bulk, the ROP polymerizations areconducted under an inert (i.e., dry) atmosphere, such as nitrogen orargon, and at a pressure of 100 MPa to 500 MPa (1 atm to 5 atm), moretypically at a pressure of 100 MPa to 200 MPa (1 atm to 2 atm). At thecompletion of the reaction, the solvent can be removed using reducedpressure.

Less preferred catalysts for the ROP polymerization include metal oxidessuch as tetramethoxy zirconium, tetra-iso-propoxy zirconium,tetra-iso-butoxy zirconium, tetra-n-butoxy zirconium, tetra-t-butoxyzirconium, triethoxy aluminum, tri-n-propoxy aluminum, tri-iso-propoxyaluminum, tri-n-butoxy aluminum, tri-iso-butoxy aluminum, tri-sec-butoxyaluminum, mono-sec-butoxy-di-iso-propoxy aluminum, ethyl acetoacetatealuminum diisopropylate, aluminum tris(ethyl acetoacetate), tetraethoxytitanium, tetra-iso-propoxy titanium, tetra-n-propoxy titanium,tetra-n-butoxy titanium, tetra-sec-butoxy titanium, tetra-t-butoxytitanium, tri-iso-propoxy gallium, tri-iso-propoxy antimony,tri-iso-butoxy antimony, trimethoxy boron, triethoxy boron,tri-iso-propoxy boron, tri-n-propoxy boron, tri-iso-butoxy boron,tri-n-butoxy boron, tri-sec-butoxy boron, tri-t-butoxy boron,tri-iso-propoxy gallium, tetramethoxy germanium, tetraethoxy germanium,tetra-iso-propoxy germanium, tetra-n-propoxy germanium, tetra-iso-butoxygermanium, tetra-n-butoxy germanium, tetra-sec-butoxy germanium andtetra-t-butoxy germanium; halogenated compound such as antimonypentachloride, zinc chloride, lithium bromide, tin(IV) chloride, cadmiumchloride and boron trifluoride diethyl ether; alkyl aluminum such astrimethyl aluminum, triethyl aluminum, diethyl aluminum chloride, ethylaluminum dichloride and tri-iso-butyl aluminum; alkyl zinc such asdimethyl zinc, diethyl zinc and diisopropyl zinc; heteropolyacids suchas phosphotungstic acid, phosphomolybdic acid, silicotungstic acid andalkali metal salt thereof; zirconium compounds such as zirconium acidchloride, zirconium octanoate, zirconium stearate, and zirconiumnitrate.

Preferred catalysts are organocatalysts whose chemical formulas containnone of the restricted metals described further above. Examples oforganocatalysts for ring opening polymerizations include tertiary aminessuch as triallylamine, triethylamine, tri-n-octylamine andbenzyldimethylamine 4-dimethylaminopyridine, phosphines, N-heterocycliccarbenes (NHC), bifunctional aminothioureas, phosphazenes, amidines, andguanidines.

A more specific organocatalyst isN-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU):

Other ROP organocatalysts comprise at least one1,1,1,3,3,3-hexafluoropropan-2-ol-2-yl (HFP) group. Singly-donatinghydrogen bond catalysts have the formula (23):R²—C(CF₃)₂OH  (23),wherein R² represents a hydrogen or a monovalent radical having 1 to 20carbons, for example an alkyl group, substituted alkyl group, cycloalkylgroup, substituted cycloalkyl group, heterocycloalkyl group, substitutedheterocycloalkyl group, aryl group, substituted aryl group, or acombination thereof. Exemplary singly-donating hydrogen bondingcatalysts are listed in Table 4.

TABLE 4

4-HFA-St

4-HFA-Tol

HFTB

NFTB

HFIP

Doubly-donating hydrogen bonding catalysts have two HFP groups,represented by the formula (24):

wherein R³ is a divalent radical bridging group comprising 1 to 20carbons, such as an alkylene group, a substituted alkylene group, acycloalkylene group, substituted cycloalkylene group, aheterocycloalkylene group, substituted heterocycloalkylene group, anarylene group, a substituted arylene group, and a combination thereof.Representative double hydrogen bonding catalysts of formula (24) includethose listed in Table 5. In a specific embodiment, R² is an arylene orsubstituted arylene group, and the HFP groups occupy positions meta toeach other on the aromatic ring.

TABLE 5

3,5-HFA-MA

3,5-HFA-St

1,3-HFAB

1,4-HFAB

In one embodiment, the catalyst is selected from the group consisting of4-HFA-St, 4-HFA-Tol, HFTB, NFTB, HPIP, 3,5-HFA-MA, 3,5-HFA-St, 1,3-HFAB,1,4-HFAB, and combinations thereof.

Also contemplated are catalysts comprising HFP-containing groups boundto a support. In one embodiment, the support comprises a polymer, acrosslinked polymer bead, an inorganic particle, or a metallic particle.HFP-containing polymers can be formed by known methods including directpolymerization of an HFP-containing monomer (for example, themethacrylate monomer 3,5-HFA-MA or the styryl monomer 3,5-HFA-St).Functional groups in HFP-containing monomers that can undergo directpolymerization (or polymerization with a comonomer) include acrylate,methacrylate, alpha, alpha, alpha-trifluoromethacrylate,alpha-halomethacrylate, acrylamido, methacrylamido, norbornene, vinyl,vinyl ether, and other groups known in the art. Examples of linkinggroups include C₁-C₁₂ alkyl, a C₁-C₁₂ heteroalkyl, ether group,thioether group, amino group, ester group, amide group, or a combinationthereof. Also contemplated are catalysts comprising chargedHFP-containing groups bound by ionic association to oppositely chargedsites on a polymer or a support surface.

The ROP reaction mixture comprises at least one organocatalyst and, whenappropriate, several organocatalysts together. The ROP catalyst is addedin a proportion of 1/20 to 1/40,000 moles relative to the cycliccarbonyl monomers, and preferably in a proportion of 1/1,000 to 1/20,000moles relative to the cyclic carbonyl monomers.

ROP Accelerators

The ROP polymerization can be conducted in the presence of an optionalaccelerator, in particular a nitrogen base. Exemplary nitrogen baseaccelerators are listed below and include pyridine (Py),N,N-dimethylaminocyclohexane (Me₂NCy), 4-N,N-dimethylaminopyridine(DMAP), trans 1,2-bis(dimethylamino)cyclohexane (TMCHD),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (−)-sparteine, (Sp)1,3-bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1),1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2),1,3-bis(2,6-di-1-propylphenyl(imidazol-2-ylidene (Im-3),1,3-bis(1-adamantyl)imidazol-2-ylidene (Im-4),1,3-di-1-propylimidazol-2-ylidene (Im-5),1,3-di-t-butylimidazol-2-ylidene (Im-6),1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-7),1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene,1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8) or acombination thereof, shown in Table 6.

TABLE 6

Pyridine (Py)

N,N-Dimethylaminocyclohexane (Me₂NCy)

4-N,N-Dimethylaminopyridine (DMAP)

trans 1,2-Bis(dimethylamino)cyclohexane (TMCHD)

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)

7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD)

1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD)

(−)-Sparteine (Sp)

1,3-Bis(2-propyl)-4,5-dimethylimidazol- 2-ylidene (Im-1)

1,3-Bis(2,4,6-trimethylphenyl)imidazol-2- ylidene (Im-2)

1,3-Bis(2,6-di-i-propylphenyl(imidazol- 2-ylidene (Im-3)

1,3-Bis(1-adamantyl)imidazol-2-yliden) (Im-4)

1,3-Di-i-propylimidazol-2-ylidene (Im-5)

1,3-Di-t-butylimidazol-2-ylidene (Im-6)

1,3-Bis(2,4,6-trimethylpheny1)-4,5- dihydroimidazol-2-ylidene (Im-7)

1,3-Bis(2,6-di-i-propylphenyl)-4,5- dihydroimidazol-2-ylidene (Im-8)

In an embodiment, the accelerator has two or three nitrogens, eachcapable of participating as a Lewis base, as for example in thestructure (−)-sparteine. Stronger bases generally improve thepolymerization rate.

The catalyst and the accelerator can be the same material. For example,some ring opening polymerizations can be conducted using1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) alone, with no another catalystor accelerator present.

The catalyst is preferably present in an amount of about 0.2 to 20 mol%, 0.5 to 10 mol %, 1 to 5 mol %, or 1 to 2.5 mol %, based on totalmoles of cyclic carbonyl monomer.

The nitrogen base accelerator, when used, is preferably present in anamount of 0.1 to 5.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2to 0.5 mol %, based on total moles of cyclic carbonyl monomer. As statedabove, in some instances the catalyst and the nitrogen base acceleratorcan be the same compound, depending on the particular cyclic carbonylmonomer.

The amount of initiator is calculated based on the equivalent molecularweight per nucleophilic initiator group. The initiator groups arepreferably present in an amount of 0.001 to 10.0 mol %, 0.1 to 2.5 mol%, 0.1 to 1.0 mol %, and 0.2 to 0.5 mol %, based on total moles ofcyclic carbonyl monomer. For example, if the molecular weight of theinitiator is 100 g/mole and the initiator has 2 hydroxyl groups, theequivalent molecular weight per hydroxyl group is 50 g/mole. If thepolymerization calls for 5 mol % hydroxyl groups per mole of cycliccarbonyl monomer, the amount of initiator is 0.05×50=2.5 g per mole ofcyclic carbonyl monomer.

In a specific embodiment, the catalyst is present in an amount of about0.2 to 20 mol %, the nitrogen base accelerator is present in an amountof 0.1 to 5.0 mol %, and the nucleophilic initiator groups of theinitiator are present in an amount of 0.1 to 5.0 mol % based on theequivalent molecular weight per nucleophilic initiator group of theinitiator.

The catalysts can be removed by selective precipitation or in the caseof the solid supported catalysts, simply by filtration. The blockcopolymer can comprise residual catalyst in an amount greater than 0 wt% (weight percent), based on total weight of the block copolymer and theresidual catalyst. The amount of residual catalyst can also be less than20 wt %, less than 15 wt %, less than 10 wt %, less than 5 wt %, lessthan 1 wt %, or most specifically less than 0.5 wt % based on the totalweight of the block copolymer and the residual catalyst.

Average Molecular Weight

The CD copolymer preferably has a number average molecular weight (Mn)as determined by size exclusion chromatography of at least 1500 g/mol,more specifically 1500 g/mol to 1,000,000 g/mol, 4000 g/mol to 150000g/mol, or 4000 g/mol to 50000 g/mol. In an embodiment, the blockcopolymer has a number average molecular weight M_(n) of 10,000 to20,000 g/mole. The block copolymer preferably has a narrowpolydispersity index (PDI) of 1.01 to 2.0, more particularly 1.01 to1.30, and even more particularly 1.01 to 1.25.

The ABA copolymer preferably has a number average molecular weight (Mn)as determined by size exclusion chromatography of at least 1500 g/mol,more specifically 1500 g/mol to 1,000,000 g/mol, 4000 g/mol to 150000g/mol, or 4000 g/mol to 50000 g/mol. In an embodiment, the blockcopolymer has a number average molecular weight M_(n) of 10,000 to20,000 g/mole. The block copolymer preferably has a narrowpolydispersity index (PDI) of 1.01 to 2.0, more particularly 1.01 to1.30, and even more particularly 1.01 to 1.25.

Endcap Agents

The ring opened polymer can further be treated with an endcap agent toprevent further chain growth and stabilize the reactive end groupsagainst unwanted side reactions such as chain scission. Endcap agentsinclude, for example, materials for converting terminal hydroxyl groupsto esters, such as carboxylic acid anhydrides, carboxylic acidchlorides, or reactive esters (e.g., p-nitrophenyl esters). In anembodiment, the endcap agent is acetic anhydride, which convertsreactive hydroxy end groups to acetate ester groups. The endcap agentcan also comprise a biologically active moiety, which becomes bound tothe terminal end group of the ring opened polymer chain.

Cytotoxicity of the Block Copolymer

The ABA and CD copolymers alone are generally non-cytotoxic. Forexample, cell viability of HepG2, a human liver carcinoma cell line, isin a range of 95% or more at CD block copolymer concentrations of 470mg/L, or higher. The cell viability of human dermal fibroblasts is alsoin the range of 95% or more at ABA TRI(35:2.4) block copolymerconcentration of 4 wt %.

Loaded Micelles

In water optionally containing organic solvent, the CD copolymerself-assembles to form a nanoparticulate micelle solution. When abiologically active cargo material is also present, the block copolymerand cargo material form a loaded micelle bound by non-covalentinteractions.

A method comprises i) forming a solution of the amphiphilic CD diblockcopolymer (i.e., carrier) and a biologically active material (i.e.,cargo) in a water miscible organic solvent, ii) dialyzing the solutionagainst deionized water using a dialysis membrane system, therebyforming an aqueous mixture comprising a loaded micelle. The loadedmicelle comprises the CD block copolymer in an amount of 85.0 wt % to99.9 wt %, and the biologically active material in an amount of about15.0 wt % to 0.1 wt %, each based on total dry weight of the loadedmicelle. Preferably, the dialysis membrane has a molecular weight cutoff(MWCU) of about 1000 Da.

The membrane dialysis can be conducted with or without agitation.Smaller and more uniform nanoparticles of the loaded micelle aregenerally favored by less mechanical agitation (e.g., stirring).Preferably, dialysis is conducted using minimal or no agitation,accompanied by occasional replacement of the external water bath withfresh deionized water, allowing the water and organic solvent toexchange by diffusion alone. The resulting aqueous mixture can becentrifuged to remove large agglomerates. The resulting supernatant cancontain about 37 wt % to about 70 wt % of the original combined dryweight of the CD copolymer and the biologically active cargo material.

The dialysis is preferably conducted for a time period of 1 hour to 5days, more preferably for a period of 1 day to 3 days.

The dialysis can be conducted at room temperature (18° C. to 28° C.), orat a lower temperature. In an embodiment, the dialysis is conducted at atemperature less than 10° C., more preferably 1° C. to 6° C. Lowertemperature dialysis favors smaller loaded micelle particle sizes.

The term “loading efficiency” refers to the percentage of the initialweight of the biologically active material that is incorporated into theloaded micelle. The loading efficiency of the biologically activematerial in the loaded micelle is preferably at least 10%. Generally,the loading efficiency of the biologically active material is in a rangeof 10% to 50%, and even more specifically in a range of 30% to 50%.

Nanoparticles of the loaded micelle can have an average particle size(circular cross sectional diameter) of 10 nm to 500 nm, 10 nm to 250 nm,and preferably 25 nm to 200 nm as measured by dynamic light scattering.For the foregoing particle sizes, the aqueous solution can have a pH of4.5 to 8.0, 5.0 to 7.0, or 6.0 to 7.0.

The organic solvent used to form a solution of the CD block copolymerand the biologically active cargo material is preferably miscible withwater at concentrations of at least 1 microliter or more of organicsolvent per 100 microliters of water. Exemplary organic solvents includemethanol, ethanol, propanol, 2-propanol, 1-butanol, 2-butanol, t-butylalcohol, acetone, 2-butanone, dimethoxyethane, diglyme, diethyl ether,methyl t-butyl ether, methylene chloride, ethyl acetate, ethyleneglycol, glycerin, dimethylsulfoxide, dimethylformamide, acetic acid,tetrahydrofuran (THF), and dioxane.

As stated above, the biologically active cargo material can be a drug.Exemplary commercially available drugs include 13-cis-Retinoic Acid,2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU,6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®,Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine,BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide,BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225,Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine,Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013,CCl-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil,Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11,Cyclophosphamide, Cyclosporin (an immunosuppressive agent, normallygiven to patients for life long after organ transplantation), Cytadren®,Cytarabine, Cytarabine Liposomal, Cytosar-U®, Cytoxan®, Dacarbazine,Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin,Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal,DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, DenileukinDiftitox, DepoCyt™, Dexamethasone, Dexamethasone Acetate, DexamethasoneSodium Phosphate Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,Doxil®, Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®,Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™, Elspar®, Emcyt®,Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-asparaginase,Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide Phosphate,Eulexin®, Everolimus, Evista®, Exemestane, Fareston®, Faslodex®,Femara®, Filgrastim, Finasteride (for hair growth), Floxuridine,Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec™,Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte—Colony StimulatingFactor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®,Herceptin®, Hexadrol, Hexylen®, Hexamethylmelamine, HMM, Hycamtin®,Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone SodiumPhosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate,Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan Idamycin®, Idarubicin,Ifex®, IFN-alpha Ifosfamide, IL-11 IL-2 Imatinib mesylate, ImidazoleCarboxamide Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, K Kidrolase (t),Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole,Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™,Liposomal Ara-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®,Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, MustineMutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AVD, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a) Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, Spironolactone,STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®,Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus,Teniposide, TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine,Thioguanine Tabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®,Toposar®, Topotecan, Toremifene, Torisel®, Tositumomab, Trastuzumab,Treanda®, Tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™,Velban®, Velcade®, VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine,Vinblastine Sulfate, Vincasar Pfs®, Vincristine, Vinorelbine,Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon®, Xeloda®,Zanosar®, Zevalin™, Zinecard®, Zoladex®, Zoledronic acid, Zolinza, andZometa.

Toxicity of the Loaded Micelle

The toxicity of the loaded micelles toward a given cell can be at leastcomparable to the toxicity of the biologically active material alonetoward the given cell. For example, a PTX loaded block copolymer can beas toxic to HepG2 as the PTX alone when tested under otherwise identicalconditions.

Formation of Composite Hydrogels

A method of preparing a composite hydrogel comprises forming a mixturecomprising a solid ABA copolymer and an aqueous loaded micelle solutioncomprising the CD copolymer and a biologically active substance, therebyforming a second mixture. Agitating the second mixture results indissolution of the ABA copolymer, thereby forming a composite hydrogel.

Another method comprises forming a first mixture comprising a loadedmicelle in water, forming a second mixture comprising the ABA copolymerin water, and combining the first mixture and the second mixture withagitation, thereby forming the composite hydrogel.

Using either of the above methods, the composite hydrogel can have asolids content of about 4 wt % to about 8 wt % based on total weight ofthe composite hydrogel.

INDUSTRIAL APPLICABILITY

The composite hydrogels can be used for human and/or non-humantherapeutic treatments. The compositions can be administered in the formof a powder, a pill, a liquid solution, paste, or a gel. Thecompositions can be used as a drug. The compositions can be administeredorally or by way of other body cavities, by injection, intravenously,and/or topically. The compositions are particularly attractive fordelivery of rigid, hydrophobic biologically active materials that havelow water solubility, such as paclitaxel.

A method comprises contacting a cell with a composite hydrogel, therebykilling the cell. In an embodiment, the cell is a cancer cell, and thebiologically active material is PTX.

A composite hydrogel can comprise a loaded micelle that encapsulates anantimicrobial material. The antimicrobial compositions can be applied toa human and/or non-human animal tissue, including mammalian and/ornon-mammalian animal tissue. The general term “animal tissue” includeswound tissue, burn tissue, skin, internal organ tissue, blood, bones,cartilage, teeth, hair, eyes, nasal surfaces, oral surfaces, other bodycavity surfaces, and any cell membrane surfaces. In an embodiment, amethod comprises contacting a microbe with an antimicrobial composition,thereby killing the microbe. The antimicrobial compositions can be usedin the form of a powder, a pill, or an aqueous mixture applied as afreely flowing liquid, spray, cream, injectable mixture, or gel. Usesinclude disinfectant washes for hands, skin, hair, bone, ear, eye, nose,throat, internal tissue, wounds, and teeth (e.g., as a mouthwash). Stillother uses include disinfectants for articles such as medical devices.Medical devices include swabs, catheters, sutures, stents, bedpans,gloves, facial masks, absorbent pads, absorbent garments, internalabsorbent devices, and insertable mechanical devices. In an embodiment,an article comprises a medical device in contact with the antimicrobialcomposition.

The antimicrobial compositions are also attractive as disinfectingagents for surfaces of articles (i.e., non-living articles) such as, forexample, building surfaces in homes, businesses, and particularlyhospitals. Exemplary home and commercial building surfaces includefloors, door surfaces, bed surfaces, air conditioning surfaces, bathroomsurfaces, railing surfaces, kitchen surfaces, and wall surfaces. Otherarticles include medical devices, cloths, garments, and non-medicalequipment. Surfaces of articles can comprise materials such as wood,paper, metal, cloth, plastic, rubber, glass, paint, leather, orcombinations thereof. In an embodiment, a method comprises contacting asurface of an article with the antimicrobial composition. In anotherembodiment, a method comprises contacting a surface of an article withan aqueous mixture of the antimicrobial composition.

The following examples demonstrate the preparation and use of compositehydrogels prepared with an ABA triblock copolymer and CD diblockcopolymer produced by organocatalytic ring-opening polymerization of asteroidal monomer.

EXAMPLES

Materials used in the following examples are listed in Table 7.

TABLE 7 ABBREVIATION DESCRIPTION SUPPLIER TMC Trimethylene CarbonateBoehringer Ingelheim DBU Diazabicyclo[5.4.0]Undec-7-Ene Sigma Aldrich TUN-Bis(3,5-Trifluoromethyl)Phenyl-N′- Prepared Cyclohexylthiourea belowChol-Cl Cholesteryl Chloroformate Sigma Aldrich mPEG-OH Mono-MethylPoly(Ethylene Glycol), Polymer number average molecular weight (Mn)Science 5K, degree of polymerization DP ~113 PEG10 Poly(EthyleneGlycol), number average Sigma Aldrich molecular weight (Mn) 10.9K,degree of polymerization DP ~226 PEG20 Poly(Ethylene Glycol), numberaverage Sigma Aldrich molecular weight (Mn) 20K, degree ofpolymerization DP ~452 PEG35 Poly(Ethylene Glycol), number average SigmaAldrich molecular weight (Mn) 35K, degree of polymerization DP ~795Bis-MPA 2,2-Bis(Hydroxymethyl)Propionic Acid Sigma Aldrich FITCFluorescein Isothiocyanate Sigma Aldrich

Unless, specifically mentioned, all materials were purchased fromSigma-Aldrich, TCI or Merck. All solvents were of analytical grade,purchased from Fisher Scientific or J. T. Baker and used as received.Trimethylene carbonate (TMC) was purchased from Boehringer Ingelheim(Ingelheim, Germany), and dried extensively by freeze drying processunder high vacuum. 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) wasdistilled from CaH₂ under dry N₂ and transferred to glove box. Beforetransferring into the glove box, monomers and other reagents (e.g.,mPEG-OH, PEG10, PEG20, and PEG35) were dried extensively byfreeze-drying under high vacuum. The cholesterol functionalizedaliphatic cyclic carbonate monomer (Chol-MTC) was prepared as describedbelow.

N-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU) was preparedas reported by R. C. Pratt, B. G. G. Lohmeijer, D. A. Long, P. N. P.Lundberg, A. Dove, H. Li, C. G. Wade, R. M. Waymouth, and J. L. Hedrick,Macromolecules, 2006, 39 (23), 7863-7871, and dried by stirring in dryTHF over CaH₂, filtering, and removing solvent under vacuum.

Nuclear Magnetic Resonance (NMR) Spectroscopy

The ¹H- and ¹³C-NMR spectra of monomers and polymers were recorded usinga Broker Avance 400 spectrometer, and operated at 400 and 100 MHzrespectively, with the solvent proton signal as the internal referencestandard.

Molecular Weight Determination by Size Exclusion Chromatography (SEC)

SEC was conducted using THF as the eluent for monitoring the polymerconversion and also for the determination of polystyrene equivalentmolecular weights of the macro-transfer agents. THF-SEC was recorded ona Waters 2695D Separation Module equipped with a Waters 2414differential refractometer and Waters HR-4E as well as HR-1 columns. Thesystem was equilibrated at 30° C. in THF, which served as the polymersolvent and eluent with a flow rate of 1.0 mL/min. Polymer solutionswere prepared at a concentration of about 3 mg/mL, and an injectionvolume of 100 microliters was used. Data collection and analysis wereperformed using the Astra software (Wyatt Technology Corporation, USA;version 5.3.4.14). The columns were calibrated with a series ofpolystyrene standards ranging from M_(P)=360 Da to M_(P)=778 kDa(Polymer Standard Service, USA).

Monomer Synthesis

Cholesteryl bearing cyclic carbonate monomer Chol-MTC was synthesizedfrom commercially available cholesteryl chloroformate (Chol-Cl)according to Scheme 1.

The preparation involves three steps: 1) reaction of the choloroformateChol-Cl with 2-bromoethyl amine hydrobromide in dichloromethane withtriethylamine (TEA) to form carbamate Chol-Br; 2) base-catalyzedreaction of Chol-Br with the acid diol bis-MPA in dimethylformamide(DMF)/KOH to form the diol ester Chol-MPA, and 3) triphosgene-mediatedcyclization of Chol-MPA to form the cyclic carbonate monomer Chol-MTC,with overall yield of about 26%. Detailed procedures of each of thethree steps are provided below. ¹H and ¹³C NMR were used to confirm thestructures of the intermediates and the cyclic carbonate monomer.

Example 1

Preparation of Chol-Br. In a 500 mL round bottom flask, equipped with amagnetic stir bar, cholesterol chloroformate (25.0 g, 55.7 mmol, 1.0equiv.) and 2-bromoethylamine hydrobromide (12.9 g, 63.0 mmol, 1.1equiv.) were suspended in dichloromethane (200 mL) and the suspensionwas chilled in an ice-bath. To this suspension, a solution oftriethylamine (TEA) (18.0 mL, 13.06 g, 129.1 mmol, 2.3 equiv.) indichloromethane (100 mL) was added dropwise over 1 hour. The reactionmixture was maintained in the bath for an additional hour and wasallowed to warm to room temperature. The reaction was then allowed toproceed for another 14 h, after which dichloromethane was removed undervaccuo and the resultant solids were suspended in a 1:1 mixture of ethylacetate and hexanes (300 mL). Organic layer was washed 2 times with amixture of saturated brine (100 mL) and de-ionized water (50 mL), andone time with saturated brine (100 mL). The organic layer was dried oversodium sulfate and the solvents were removed under vacuuo to yield apale yellow solid (29.1 g, 97.4%). As the crude product was determinedto have satisfactory purity by ¹H NMR, no further purification wasconducted. ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.38 (CH═C incholesterol), 5.03 (NHCOO of side-chain), 4.50 (CH—OCONH ofcholesterol), 3.58 (BrCH₂CH₂NH), 2.45-0.6 (the remaining protons fromcholesterol).

Example 2

Synthesis of Chol-MPA. In a 500 mL round bottom flask with magnetic stirbar, a mixture of KOH (85%, 2.0 g, 30.3 mmol, 1.1 equiv.), bis-MPA (4.20g, 31.3 mmol, 1.1 equiv.) and dimethylformamide (DMF) (200 mL) wereheated to 100° C. for 1.5 hours. A homogenous solution was formed, andChol-Br (15.0 g, 28.0 mmol, 1.0 equiv.) was added to the hot solution.Stirring was continued with heating for 16 hours and most of the DMF wasremoved under reduced pressure, to result in oily semisolid, which wasthen dissolved in 2:1 ethyl acetate:hexanes mixture (300 mL). Theorganic solution was washed with saturated brine (100 mL) and de-ionizedwater (100 mL) mixture. The resultant aqueous layer was extracted withethyl acetate (3×100 mL) to recover Chol-MPA lost during the washingprocess. The combined organic layers were washed with saturated brine(80 mL) and de-ionized water (20 mL) mixture. The combined organic layerwas dried with Na₂SO₄ and the solvent removed in vacuuo to result incrude product as a pale white waxy solid (16.5 g). The crude product waspurified by flash column chromatography using silica as the packingmaterial and a gradient of hexanes to ethyl acetate as the eluent toresult in the final product Chol-MPA as a waxy white solid (10.7 g,64.8%). ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.35 (CH═C in cholesteroland NHCOO of side-chain), 4.47 (CH—OCONH of cholesterol), 4.26(CH₂CH₂NHCOO), 3.88 and 3.72 (CH₂OH) 3.45 (CH₂CH₂NHCOO), 3.34 (OH),2.50-0.60 (the remaining protons from cholesterol and CH₃ from bis-MPA).

Example 3

Preparation of Chol-MTC. In a 500 mL round bottom flask with magneticstir bar, Chol-MPA (10.1 g, 17.1 mmol, 1.0 equiv.) was dissolved inanhydrous dichloromethane (150 mL). Pyridine (8.2 mL, 8.0 g, 101.5 mmol,5.9 equiv.) was added and the solution was cooled in a dry ice-acetonebath (−78° C.). To this cooled reaction mixture, triphosgene (2.69 g,9.06 mmol, 1.9 equivalents based on functional equivalents oftriphosgene) solution (dissolved in 50 mL dichloromethane) was addeddropwise over 1 hour. After 1 hour, from −78° C., the reaction mixturewas allowed to warm up to room temperature, and after 2 hours, thereaction was quenched by adding saturated aqueous ammonium chloridesolution (50 mL). The organic layer was washed twice with a mixture of1.0 N HCl (20 mL) and saturated brine (80 mL), followed by a mixture ofsaturated brine (50 mL) and saturated NaHCO₃ (50 mL), dried usingNa₂SO₄. Removal of solvent in vacuo resulted in crude product as aslightly yellowish solid. The crude product was further purified byflash column chromatography using silica as the packing material and agradient of chloroform to chloroform:ethyl acetate(4:1) mixtures as theeluent, to result in the final product Chol-MTC as a waxy white solid(6.8 g, 65%). ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.35 (CH═C incholesterol), 4.95 (NHCOO), 4.86 and 4.27 (CH₂OCOOCH₂), 4.47 (CH—OCONHof cholesterol), 4.27 (CH₂CH₂NHCOO), 3.45 (CH₂CH₂NHCOO), 2.40-0.60 (theremaining protons from cholesterol and CH₃ in the cyclic carbonatemonomer).

Preparation of CD Diblock Copolymers by Ring Opening Polymerization

The ring opening homopolymerization of Chol-MTC resulted in lowconversion and/or low degree of polymerization when the ring openingpolymerization was initiated by benzyl alcohol (BzOH) or mPEG-OH. On theother hand, copolymerization of Chol-MTC with TMC was found to occurwith high conversion (>90%). Therefore, TMC was used as a diluentcomonomer to prepare several amphiphilic block copolymers according toScheme 2.

The TMC not only assists in achieving high monomer conversions, but alsoserves to dilute/spread out the rigid cholesteryl side chain groupsacross the hydrophobic region. The cholesteryl bearing polymers preparedaccording to Scheme 2 are represented by the name Polymer(x:y), where xand y represent numbers of units derived from Chol-MTC and TMC,respectively, in the block copolymer structure of Scheme 2. Thesubscript n in the mPEG-OH structure (Scheme 2) was about 113,corresponding to a number average molecular weight (Mn) of 5000 Da. Thestacked subunit structures between the vertical brackets in the blockcopolymer structure of Scheme 2 indicate the polycarbonate block ofPolymer(x:y) is a random copolymer of repeat units derived from Chol-MTCand TMC.

In the following Examples 4 to 9, amounts were calculated based onfollowing: i) the initiator (mPEG-OH) has one nucleophilic initiatinggroup (hydroxy group) per mole of the initiator, ii) the molecularweight of mPEG-OH=5000 g/mole, and iii) the molar amount of initiatorwas set to one equivalent relative to equivalents of cyclic carbonylmonomer.

Example 4

Preparation of Polymer(0:67). In a 7 mL vial containing a magnetic stirbar, in glove box, TMC (MW=102.1, 922 mg, 9.03 mmol, 180.6 equivalents),mPEG-OH (5 kDa, 250 mg, 50.0 micromoles, 1.0 equivalent) and TU(MW=370.4, 47.0 mg, 127 micromoles, 2.5 equiv.) were dissolved indichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7microliters, 19.1 mg, 124 micromoles, 2.5 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andsize exclusion chromatography (SEC). After 40 hours, the reaction wasquenched by the addition of about 20 mg of benzoic acid and wasprecipitated in ice-cold diethyl ether (2×50 mL). Polymer(0:67) wasdried in a tared vial for about 1 to 2 days, until a constant samplemass was obtained as white powder (580 mg). The number average molecularweight by NMR (Mn (NMR))=11800 Da. ¹H NMR (400 MHz, CDCl₃, delta, ppm):4.20-4.30 (CH₂CH₂CH₂OCOO), 3.85-3.5, 3.38 (CH₃ of mPEG), 2.10-2.00(CH₂CH₂CH₂OCOO). The degree of polymerization (DP) of TMC was 67. Thepolydispersity index (PDI) was 1.12.

Example 5

Preparation of Polymer(4:0). In a 7 mL vial containing a magnetic stirbar, in glove box, Chol-MTC (MW=616, 312 mg, 507 micromoles, 10.1equivalents), mPEG-OH (5 kDa, 251 mg, 50.2 micromoles, 1.0 equivalent)and TU (MW=370.4, 46.0 mg, 125 micromoles, 2.5 equiv.) were dissolved indichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7microliters, 19.1 mg, 124 micromoles, 2.5 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andSEC. After 18 hours, the reaction was quenched by the addition of about20 mg of benzoic acid and was precipitated in ice-cold diethyl ether(2×50 mL). Polymer (4:0) was dried in a tared vial for about 1 to 2days, until a constant sample mass was obtained as white powder (356mg). Mn (NMR)=7500 Da. ¹H NMR (400 MHz, CDCl₃, δ, ppm): 5.45-5.35 (CH═Cin cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH ofcholesterol), 4.40-4.10 (CH₂OCOOCH₂ and CH₂CH₂NHCOO), 3.85-3.5 (CH₂CH₂Oof mPEG) 3.5-3.30 (CH₂CH₂NHCOO), 3.38 (CH₃ of mPEG), 2.45-0.55 (rest ofthe protons from cholesterol and CH₃ in the cyclic carbonate monomer).The DP of Chol-MTC was 4. The PDI was 1.12.

Example 6

Preparation of Polymer(11:0). In a 7 mL vial containing a magnetic stirbar, in glove box, Chol-MTC (MW=616, 312 mg, 995 micromoles, 19.1equivalents), mPEG-OH (5 kDa, 260 mg, 52.0 micromoles, 1.0 equivalent)and TU (MW=370.4, 48.0 mg, 130 micromoles, 2.5 equiv.) were dissolved indichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7microliters, 19.1 mg, 124 micromoles, 2.4 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andSEC. After 6 hours, the reaction was quenched by the addition of about20 mg of benzoic acid and was precipitated in ice-cold diethyl ether(2×50 mL). Polymer (11:0) was dried in a tared vial for about 1 to 2days, until a constant sample mass was obtained as white powder (612mg). Mn (NMR)=11800 Da. ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.45-5.35(CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40(CH—OCONH of cholesterol), 4.40-4.10 (CH₂OCOOCH₂ and CH₂CH₂NHCOO),3.85-3.5 (CH₂CH₂O of mPEG) 3.5-3.30 (CH₂CH₂NHCOO), 3.38 (CH₃ of mPEG),2.45-0.55 (rest of the protons from cholesterol and CH₃ in the cycliccarbonate monomer). The DP of Chol-MTC was 11. PDI was 1.21.

Ring opened block copolymers having different x:y ratios (Chol-MTCunits:TMC units) were prepared using the initiator mPEG-OH (5.0 kDa).

Example 7

Preparation of Polymer (8:8). In a 7 mL vial containing a magnetic stirbar, in glove box, Chol-MTC (MW=616, 617 mg, 1000 micromoles, 9.9equivalents), TMC (MW=102.1, 109 mg, 1.07 mmol, 10.6 equivalents),mPEG-OH (5 kDa, 506 mg, 101 micromoles, 1.0 equivalent) and TU(MW=370.4, 103 mg, 278 micromoles, 2.7 equiv.) were dissolved indichloromethane (2.0 mL). To this solution, DBU (MW=152.24, 37.3microliters, 38.0 mg, 250 micromoles, 2.5 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andSEC. After 5 hours, the reaction was quenched by the addition of about30 mg of benzoic acid and was precipitated in ice-cold diethyl ether(2×50 mL). Polymer (8:8) was dried in a tared vial for about 1 to 2days, until a constant sample mass was obtained as white powder (968mg). Mn (NMR)=10700 Da. ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.45-5.35(CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40(CH—OCONH of cholesterol), 4.40-4.10 (CH₂OCOOCH₂, CH₂CH₂NHCOO andCH₂CH₂CH₂OCOO of TMC), 3.85-3.5 (CH₂CH₂O of mPEG) 3.5-3.30(CH₂CH₂NHCOO), 3.38 (CH₃ of mPEG), 2.45-0.55 (rest of the protons fromcholesterol, CH₃ in the cyclic carbonate monomer and CH₂CH₂NHCOO ofTMC). The DP of Chol-MTC was 8. The DP of TMC was 8. The PDI was 1.18.

Example 8

Preparation of Polymer(11:30). In a 7 mL vial containing a magnetic stirbar, in glove box, Chol-MTC (MW=616, 699 mg, 520 micromoles, 10.4equivalents), TMC (MW=102.1, 154 mg, 1.51 mmol, 30.3 equivalents),mPEG-OH (5 kDa, 249 mg, 49.8 micromoles, 1.0 equivalent) and TU(MW=370.4, 46.0 mg, 125 micromoles, 2.5 equiv.) were dissolved indichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7microliters, 19.1 mg, 124 micromoles, 2.5 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andSEC. After 18 hours, the reaction was quenched by the addition of about20 mg of benzoic acid and was precipitated in ice-cold diethyl ether(2×50 mL). Polymer (11:30) was dried in a tared vial for about 1 to 2days, until a constant sample mass was obtained as white powder (614mg). Mn (NMR)=14800 Da. ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.45-5.35(CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40(CH—OCONH of cholesterol), 4.40-4.10 (CH₂OCOOCH₂, CH₂CH₂NHCOO andCH₂CH₂CH₂OCOO of TMC), 3.85-3.5 (CH₂CH₂O of mPEG) 3.5-3.30(CH₂CH₂NHCOO), 3.38 (CH₃ of mPEG), 2.45-0.55 (rest of the protons fromcholesterol, CH₃ in the cyclic carbonate monomer and CH₂CH₂NHCOO ofTMC). DP of Chol-MTC was 11. The DP of TMC was 30. The PDI was 1.20.

Example 9

Preparation of Polymer(18:55). In this example, a higher level ofcatalyst (5.0 equiv of with respect to initiator) was used because itwas found from Example 4 that polymerization kinetics were slower forrelatively high initiator to monomer ratios. In a 7 mL vial containing amagnetic stir bar, in glove box, Chol-MTC (MW=616, 876 mg, 1.42 mmol,19.6 equivalents), TMC (MW=102.1, 452 mg, 4.43 mmol, 61.2 equivalents),mPEG-OH (5 kDa, 362 mg, 72.4 micromoles, 1.0 equivalent) and TU(MW=370.4, 134 mg, 362 micromoles, 4.8 equiv.) were dissolved indichloromethane (3.0 mL). To this solution, DBU (MW=152.24, 52.3microliters, 53.3 mg, 350 micromoles, 5.0 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andSEC. After 4 hours, the reaction was quenched by the addition of about30 mg of benzoic acid and was precipitated in ice-cold diethyl ether(2×50 mL). Polymer (18:55) was dried in a tared vial for about 1 to 2days, until a constant sample mass was obtained as white powder (533mg). Mn (NMR)=21700 Da. ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.45-5.35(CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40(CH—OCONH of cholesterol), 4.40-4.10 (CH₂OCOOCH₂, CH₂CH₂NHCOO andCH₂CH₂CH₂OCOO of TMC), 3.85-3.5 (CH₂CH₂O of mPEG) 3.5-3.30(CH₂CH₂NHCOO), 3.38 (CH₃ of mPEG), 2.45-0.55 (rest of the protons fromcholesterol, CH₃ in the cyclic carbonate monomer and CH₂CH₂NHCOO ofTMC). DP of Chol-MTC was 18. The DP of TMC was 55. The PDI was 1.17.

Example 10

Preparation of Polymer(29:77). In a 7 mL vial containing a magnetic stirbar, in glove box, Chol-MTC (MW=616, 946 mg, 1.54 mmoles, 30.0equivalents), TMC (MW=102.1, 464 mg, 4.55 mmol, 92.4 equivalents),mPEG-OH (5 kDa, 256 mg, 51.2 micromoles, 1.0 equivalent) and TU(MW=370.4, 95.0 mg, 257 micromoles, 4.9 equiv.) were dissolved indichloromethane (3.0 mL). To this solution, DBU (MW=152.24, 37.3microliters, 38 mg, 250 micromoles, 5.0 equivalents) was added toinitiate polymerization. The reaction mixture was allowed to stir atroom temperature and aliquots of samples were taken to monitor themonomer conversion and the molecular weight by ¹H NMR spectroscopy andSEC. After 4 hours, the reaction was quenched by the addition of about30 mg of benzoic acid and was precipitated in ice-cold diethyl ether(2×50 mL). Polymer (29:77) was dried in a tared vial for about 1 to 2days, until a constant sample mass was obtained as white powder (590mg). Mn (NMR)=30700 Da. ¹H NMR (400 MHz, CDCl₃, delta, ppm): 5.45-5.35(CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40(CH—OCONH of cholesterol), 4.40-4.10 (CH₂OCOOCH₂, CH₂CH₂NHCOO andCH₂CH₂CH₂OCOO of TMC), 3.85-3.5 (CH₂CH₂O of mPEG) 3.5-3.30(CH₂CH₂NHCOO), 3.38 (CH₃ of mPEG), 2.45-0.55 (rest of the protons fromcholesterol, CH₃ in the cyclic carbonate monomer and CH₂CH₂NHCOO ofTMC). The DP of Chol-MTC was 29. The DP of TMC was 77. The PDI was 1.21.

Each of the above described block copolymers has a polydispersity index(PDI) of less than 1.25.

Properties of the self-assembled polymers without drug loading aresummarized in Table 8. Example 4 and Examples 7 to 10 have an x:y ratioless than or equal to 1.0 (x:y=0:67, 8:8, 11:30; 18:55; 29:77,respectively). Examples 5 and 6 have an x:y ratio greater than 1.0(x:y=4:0, 11:0, respectively).

TABLE 8 Units of Units of Micelle Chol-MTC TMC CMC Size ExamplePolymer(x:y) (x) (y) Mn (mg/L) (nm) Micelle PDI 4 Polymer(0:67) 0 6711800 2.1 163 ± 4  0.56 ± 0.01 5 Polymer(4:0) 4 0 7500 — 234.5 ± 33.00.49 ± 0.00 6 Polymer(11:0) 11 0 11600 2.1 114 ± 1  0.14 ± 0.01 7Polymer(8:8) 8 8 10700 1.5 133 ± 3  0.44 ± 0.01 8 Polymer(11:30) 11 3014800 1.5 36 ± 1 0.08 ± 0.05 9 Polymer(18:55) 18 55 21700 2.1 229 ± 510.29 ± 0.02 10 Polymer(29:77)^(a) 29 77 30700 ^(a)Example 10 was notfurther characterized or used for preparing loaded micelles below due toits hydrophobicity.Critical Micelle Concentrations of CD Copolymers

General procedure for polymer self-assembly. The following dialysismethod was used to prepare non-loaded micelles of the polymers ofExamples 4 to 9. In a scintillation vial (20 mL) with a magnetic stirbar, block copolymer (15.0 mg) was dissolved in DMF (2.0 mL) withstirring at room temperature for about 1 hour to 2 hours. The polymersolution in DMF was then transferred to a prewashed dialysis membranehaving a molecular weight cutoff (MWCO) of 1000 Da (Spectra/Por) and wasdialyzed at 4° C. against deionized water (1 L). The water was changedat 3, 6 and 24 hours. At the end of the dialysis process, the resultingmicelle solution was centrifuged at 4000 rpm for 5 minutes to removelarge aggregates. Typically the final concentration of the polymersolution after dialysis was about 1.0 mg/mL.

Fluorescence Measurements. The critical micelle concentrations (CMCs) ofthe polymers in DI water were determined by fluorescence spectroscopyusing pyrene as the probe. The fluorescence spectra were recorded by anLS 50B luminescence spectrometer (Perkin Elmer, U.S.A.) at 25° C.Dialyzed polymer samples (see below) were equilibrated for 10 min beforetaking measurements. Aliquots of pyrene in acetone solution (6.16×10⁻⁵M, 10 microliters) were added to glass vials and air dried to remove theacetone. Polymer solutions of varying concentrations were added to thepyrene at 1 mL each, and left to stand for 24 hours. The final pyreneconcentration in each vial is 6.16×10⁻⁷ M. The excitation spectra werescanned at wavelength from 300 nm to 360 nm with an emission wavelengthof 395 nm. Both the excitation and emission bandwidths were set at 2.5nm. The intensity (peak height) ratio of 1339/1334 from the excitationspectra was analyzed as a function of polymer concentration. The CMC wastaken at the point of intersection between the tangent to the curve atthe inflection and tangent of the points at low concentrations.

Transmission electron microscopy. The morphologies of the polymers underaqueous conditions were observed under a FEI Tecnai G2 F20 electronmicroscope using an acceleration voltage of 200 keV. The TEM sampleswere prepared by first placing a drop of aqueous polymer solution (4.0microliters) onto a formvar coated 200 mesh copper grid (Ted Pella Inc.,USA). After 1 min, the excess solution was wicked off by using filterpaper. Then the staining agent of phosphotungstic acid (2% w/v; 4.0microliters) was placed on the grid and after a minute, the excesssolution was wicked off and the grid was left to dry under ambientconditions.

FIG. 1 is a graph showing the intensity (peak height) ratio of 1339/1334of the excitation spectra as a function of polymer concentration forExample 4 (x:y=0:67), Example 6 (x:y=11:0), Example 7 (x:y=8:8), Example8 (x:y=11:30), and Example 9 (x:y=18:55). The CMC values in DI water ofExamples 4, and 6 to 9 are, respectively, 2.1 mg/L, 2.1 mg/L, 1.5 mg/L,1.5 mg/L and 2.1 mg/L (Table 8). The CMC values of the polymers ofExample 5 and Example 10 were not determined. The low CMC values ofExamples 6 to 8 indicate that the micellar structure can exist even atlow concentrations, thereby enabling in vivo application of thesematerials where the micellar solution would undergo an extensivedilution after administration. The polymers are expected to exhibit evenlower CMC values in the bloodstream as a result of the “salting out”effect.

The average particle size diameter (in nanometers) and size distribution(PDI) of non-loaded micelles formed with the polymers are also listed inTable 8. Example 8, which has an x:y ratio (Chol-MTC:TMC=11:30) lessthan 1.0 and a molecular weight less than or equal to 15 kDa, theaverage particle size was less than 40 nm. This value is significantlysmaller than the micelle particle sizes obtained with Example 5 (163nm), Example 6 (114 nm), and Example 7 (133 nm), which have an x:y ratiogreater than or equal to 1.0. The micelle polydispersity index (PDI)also indicates that the nanoparticles formed from the polymers having anx:y ratio less than 1:1 and lower molecular weight of about 15 kDa(Examples 8) have a lower particle size distribution compared to thepolymers having an x:y ratio greater than or equal to about 1.0(Examples 5 to 7).

FIGS. 2A to 2F are TEM images of the micelles formed by Example 4(x:y=0:67), Example 5 (x:y=4:0), Example 6 (x:y=11:0), Example 7(x:y=8:8), Example 8 (x:y=11:30), and Example 9 (x:y=18:55),respectively. Comparing the TEMs of Examples 4 to 6 (FIGS. 2A to 2C),self-assembly was promoted by increasing the content of cholesterylrepeat units in the block copolymer. When TMC was used as a comonomer,discrete nanostructures were observed (Examples 8 and 9, FIGS. 2D and2E, respectively). At high TMC content, predominantly collapsed vesiclesformed (Example 9, FIG. 2F). The upper image in FIG. 2F is at amagnification of 28000×. The lower image in FIG. 2F is at amagnification of 110000×.

Cytotoxicity of CD Diblock Copolymers

HepG2 cells were maintained in DMEM growth medium supplemented with 10%FBS (fetal bovine serum), 100 microgram/mL penicillin and 100 units/mLstreptomycin at 37° C., under the atmosphere of 5% CO₂. To assess thecytotoxicity of the amphiphilic block copolymer (Examples 4, andExamples 6 to 9) in HepG2 cells, a standard MTT (dimethyl thiazolyldiphenyl tetrazolium salt) assay protocol was employed. On a 96-wellplate, cells were seeded at a density of 1×10⁴ cells/well and allowed togrow for 24 hours to reach 60% to 70% confluence. Each well was replacedwith 100 microliters of fresh growth medium and treated with 10microliters of the block copolymer solution. The cytotoxicity test wasperformed in replicates of 6 wells per block copolymer or PTXconcentration. After 4 hours of incubation, the wells were replaced withfresh medium and incubated further for 68 hours. Upon replacing thewells with 100 microliters of fresh medium and 20 microliters of MTTsolution (5 mg/mL in PBS buffer), the cells were incubated for another 4hours. Finally, the used media were removed and the internalized purpleformazan crystals in each well were dissolved with 150 microliters ofDMSO. A 100 microliter aliquot of the formazan/DMSO solution wastransferred from each well to a new 96-well plate, and the absorbance(A) was measured using a microplate spectrophotometer (BioTekInstruments Inc, Winooski, Vt., U.S.A.) at the wavelength of 550 nm and690 nm. To measure the relative cell viability in different polymer, theabsorbance of formazan solution in the treated cells were compared tothat of the control cells:Cell viability=[(A ₅₅₀ −A ₆₉₀)sample/(A ₅₅₀ −A ₆₉₀)control]×100%where A₅₅₀ and A₆₉₀ represent absorbance at 550 nm and 690 nm,respectively, (A₅₅₀-A₆₉₀)sample represents the difference in theabsorbance at 550 nm and 690 nm of the sample, and (A₅₅₀-A₆₉₀)controlrepresents the difference in the absorbance at 550 nm and 690 nm for thecontrol.

The data were statistically analyzed for significant differences, basedon the Student's t-test at p<0.05. FIG. 3 is a graph showing HepG2 cellviability (%) against block copolymer concentration (Examples 4, and 6to 9) without the drug. Each of the block copolymers was non-cytotoxic.Greater than 90% cell viability was observed even at the highest polymerconcentration (2377 mg/L).

Preparation of Drug Loaded Micelles

The following examples demonstrate the following drugs can be physicallyentrapped by non-covalent interactions in the self-assembled diblockcopolymer: Paclitaxel (PTX), Cyclosporin A (CYC, an immunosuppressiveagent used after organ transplantation) and Spironolactone (SPL, a drugused to treat hypertension). The drugs can be used singularly or incombination.

General procedure for all three drugs (PTX, CYC and SPL). The dialysismethod was used to prepare loaded micelles with the drugs. Polymer (15mg) and drug (3 mg) were dissolved together in 2 mL DMF in theirrespective ratios. The mixture was placed in a dialysis membrane bagwith molecular weight cut-off (MWCO) of 1000 Da (Spectrum Laboratories,U.S.A.). The dialysis bag was then immersed in 1 L of deionized (DI)water at 4° C. without stirring for 2 days. During the 2-day course, theexternal dialysis medium was replaced at 3 hours, 6 hours and 24 hours.At the end of the dialysis process, the resulting micelle solution wascentrifuged at 4000 rpm for 5 minutes to remove large agglomerates. Thedry weight of the agglomerates was about 30 wt % to about 60 wt % of thecombined dry weight of the polymer and drug. The loaded micelles werecharacterized with respect to their size with a Zetasizer with dynamiclight scattering capability (scattering angle: 90°) and equipped with aHe—Ne laser beam at 658 nm (Malvern Instruments Zetasizer Nano ZS, UK).

PTX loading measurements. To determine the encapsulation efficiency andloading level of PTX, the drug-loaded micelles were first freeze-driedand then re-dissolved in 200 microliters of dichloromethane. 6 mL ofcold ether was then added to precipitate the polymer. The mixture wascentrifuged at 4000 rpm for 20 minutes and the supernatant wastransferred into a fresh tube, and air dried. The deposited drug wasdissolved in 4 ml, of mobile phase consisting ofwater:methanol:acetonitrile in the volume ratio of 35:20:45, andfiltered through a 0.22 micrometer filter to remove any largeaggregates. PTX loading level as weight percent (wt %) based on thetotal weight of the dry loaded micelle was analyzed using highperformance liquid chromatography (HPLC, Waters 996 PDA detector,U.S.A.) at 228 nm. The drug loading efficiency (i.e., the percentage ofthe initial amount in milligrams of drug that is successfullyencapsulated) was also reported. For example, if the loading efficiencyis 40%, then 40% of the initial amount of paclitaxel was incorporatedinto the loaded micelles obtained after freeze-drying.

CYC loading measurements. To determine the encapsulation efficiency andloading level of CYC, the drug-loaded micelles were first freeze-driedand then re-dissolved in 200 microliters of dichloromethane. 6 mL ofcold ether was then added to precipitate the polymer. The mixture wascentrifuged at 4000 rpm for 20 minutes and the supernatant wastransferred into a fresh tube, and air dried. The deposited drug wasdissolved in 4 ml, of mobile phase consisting of water:acetonitrile inthe volume ratio of 20:80. CYC loading level as weight percent (wt %)based on the total weight of the drug-loaded micelle was analyzed usinghigh performance liquid chromatography (HPLC, Waters 996 PDA detector,U.S.A.) at 210 nm.

SPL loading measurements. To determine the encapsulation efficiency andloading level of SPL, the drug-loaded micelles were first freeze-driedand then re-dissolved in 200 microliters of dichloromethane. 6 mL ofcold ether was then added to precipitate the polymer. The mixture wascentrifuged at 4000 rpm for 20 minutes and the supernatant wastransferred into a fresh tube, and air dried. The deposited drug wasdissolved in 4 ml, of mobile phase consisting of water:acetonitrile inthe volume ratio of 50:50. SPL loading level as weight percent (wt %)based on the total weight of the drug-loaded micelle was analyzed usinghigh performance liquid chromatography (HPLC, Waters 996 PDA detector,U.S.A.) at 238 nm.

Examples 11 to 16

Using the above general procedure, drug loaded micelles were preparedusing the polymers of Examples 4, and 6 to 10 by dialyzing at 4° C.without stirring. The loaded micelles are designated by the namePTX-Polymer(x:y), CYC-Polymer(x:y) and SPL-Polymer(x:y), where x is thenumber of repeat units derived from Chol-MTC and y is the number ofrepeat units derived from TMC.

The effects of block copolymer structure and initial drug loading on theparticle size, polydispersity index (PDI), final drug loading level, andloading efficiency of the PTX loaded micelles are shown in Tables 9, 10and 11, respectively.

TABLE 9 Units of Units Loaded Chol- of Micelle Loaded Loading LoadingInitial PTX MTC TMC Size Micelle level efficiency Example Loaded Micelle(mg) (x) (y) (nm) PDI (wt %) (%) 11 PTX- 3 0 67 142 ± 52 0.49 ± 0.17 5.0± 2.7 27.6 ± 7.8 Polymer(0:67) 12 PTX- 3 11 0 115 ± 9  0.13 ± 0.03 3.8 ±0.3 23.3 ± 3.6 Polymer(11:0) 13 PTX- 3 8 8 131 ± 9  0.45 ± 0.08 9.2 ±0.8 38.8 ± 5.0 Polymer(8:8) 14 PTX- 3 11 30 36 ± 1 0.07 ± 0.01 15.0 ±1.8  56.8 ± 0.3 Polymer(11:30) 15 PTX- 3 18 55 200 ± 44 0.22 ± 0.03 8.4± 2.4 25.5 ± 0.3 Polymer(18:55)

TABLE 10 Units of Units Loaded Initial Chol- of Micelle Loaded LoadingLoading CYC MTC TMC Size Micelle level efficiency Example Loaded Micelle(mg) (x) (y) (nm) PDI (wt %) (%) 16 CYC- 3 0 30 125 ± 18  0.39 ± 0.02 7.9 ± 1.6 31.9 ± 3.8 Polymer(0:30) 17 CYC- 3 11 0 123 ± 0.3 0.20 ± 0.0112.7 ± 0.4 51.3 ± 4.7 Polymer(11:0) 18 CYC- 3 11 30  36 ± 0.2 0.04 ±0.01 16.8 ± 2.6 60.6 ± 3.7 Polymer(11:30)

TABLE 11 Units of Units Loaded Initial Chol- of Micelle Loaded LoadingLoading CYC MTC TMC Size Micelle level efficiency Example Loaded Micelle(mg) (x) (y) (nm) PDI (wt %) (%) 19 SPL- 3 0 30 112 ± 24 0.43 ± 0.01 3.7± 0.3 11.9 ± 1.6 Polymer(0:30) 20 SPL- 3 11 0 102 ± 2  0.16 ± 0.01 4.3 ±0.2 14.3 ± 0.9 Polymer(11:0) 21 SPL- 3 11 30 37 ± 1 0.08 ± 0.02 4.9 ±1.1 14.4 ± 6.4 Polymer(11:30)

Overall, an initial amount of PTX of 3 mg produced loaded micelleshaving a particle size (i.e., average circular cross-sectional diameter)in a range of 36 nm to 200 nm (Examples 11 to 15, Table 9). Theoptimization studies of the fabrication conditions for the loadedmicelle show that stirring conditions had less influence on the particlesize than the temperature at which the loaded micelles were prepared.Hence, the particles were formed solely based on slow diffusion andsolvent exchange. Particle sizes less than 200 nm are attractive fordrug delivery because the particles are less susceptible to clearance bythe reticuloendothelial system (RES), and have a more favorablebiodistribution into tumor tissues through the enhanced permeability andretention (EPR) effect. This passive targeting phenomenon is attributedto two factors: the disorganized and leaky tumor vasculature which leadsto hyperpermeability to macromolecules, and at the same time, tumortissues have poor lymphatic drainage, which prevents escape of thesemacromolecules after entry. In greater detail, tumor vessels are highlydisorganized, defective or leaky having gap sizes of 100 nm to 2micrometers. This allows nanoparticles to escape easily from the bloodstream and accumulate within tumors. Furthermore, re-entry back into thebloodstream is prevented by the defective lymphatic drainage in thetumor.

Under the same fabrication conditions, copolymers prepared from amixture of Chol-MTC and TMC (Examples 13 to 15, Table 9) hadsignificantly higher drug loading capacities compared to the loadingcapacities of the block copolymer formed with TMC alone (Example 11,PTX-Polymer(0:67)), or the block copolymer prepared with Chol-MTC alone(Example 12, PTX-Polymer(11:0)). The combination of TMC and Chol-MTC inthe block copolymer provided increased loading efficiency (%),especially at higher x:y ratios (compare Examples 11 and 12 to Examples13 and 14, Table 9). Lower PTX loading level (wt %), lower loadingefficiency (%), and larger particle size were observed with Example 15(PTX-Polymer(18:55)), attributed to precipitation of the polymer and/orPTX during the dialysis process.

FIGS. 4A to 4E are TEM images of loaded micelles formed by Example 11(PTX-Polymer(0:67), FIG. 4A), Example 12 (PTX-Polymer(11:0), FIG. 4B),Example 13 (PTX-Polymer(8:8), FIG. 4C), Example 14 (PTX-Polymer(11:30),FIG. 4D), and Example 15 (PTX-Polymer(18:55), FIG. 4E), respectively.When Chol-MTC was used alone in the ring opening polymerization (Example12, FIG. 4B), the resulting block copolymer self-assembled intocylindrical columns about 20 nanometers wide. The columns comprised astack of discs, each disc having a height of about 5 nanometers; thus, astack of 9 discs corresponds to a cylindrical column length of about 45nanometers. When Chol-MTC was copolymerized with TMC, discrete sphericalnanostructures were formed (Examples 13 to 15, FIGS. 4C to 4E,respectively) having an average diameter of about 50 nm to about 100 nm.

In Vitro Release Studies

Release of drugs from the drug-loaded nanoparticles (Examples 11 to 15)was studied using the dialysis method. A dialysis membrane tube withMWCO of 2000 Da (Spectrum Laboratories, U.S.A.) containing 2 ml of themicelles was immersed in 40 ml of the release medium (i.e., PBS at pH7.4) containing 0.1% (v/v) TWEEN 80 (sold by ICI Americas, Inc.) tomaintain a sink condition. This was kept shaking on an orbital shaker at120 rpm at 37° C. At designated time intervals (1, 7, 24, 31, 48, 55,120 and 144 hours), the release medium was removed and replaced withfresh medium. The collected medium was analyzed for its drug content. Todo this, 10 ml of dichloromethane (DCM) was added and mixed with therelease medium through 3 minutes of vigorous mixing. The organic layerwas allowed to settle and was carefully extracted into a new vial. DCMwas evaporated by air flow. The deposited drug was dissolved in 4 ml ofmobile phase consisting of 20 mM ammonium acetate inwater:methanol:acetonitrile in the volume ratio of 35:20:45. Drugcontent was analyzed using high performance liquid chromatography (HPLC,Waters 996 PDA detector, U.S.A.) at 228 nm UV wavelength.

FIGS. 5A to 5C are graphs showing the cumulative release of PTX, CYC andSPL, respectively, with time. Approximately 100% of the PTX was releasedfrom PTX-Polymer(11:0) micelles (Example 12, FIG. 5A) within 24 hours.About 60% of the PTX was released by 48 hours, and was sustained formore than 6 days from the remaining PTX loaded micelles (Examples 11,13, 14 and 15, FIG. 5A). For CYC, drug release was sustained from themicellar formulation (CYC-Polymer(11:30), Example 18) for about 5 days(FIG. 5B). SPL release from the micellar formulation(SPL-Polymer(11:30), Example 21) was sustained for about 7 hours (FIG.5C).

Killing Efficiency of Tumor Cells by PTX-Loaded Micelles

Killing efficiency of HepG2 cells by PTX-loaded micelles (Examples 11 to15) was studied according to the protocol described above for the blockcopolymers alone. FIG. 6 is a bar chart comparing viability of HepG2cells after incubation for 48 hours with PTX loaded micelles (Examples11 to 15) and free PTX (PTX not bound to a polymer) at various PTXconcentrations. The PTX loaded micelles had better killing efficiencythan the free PTX alone at all PTX levels. As described above (see alsoFIG. 3) the polymers alone were not cytotoxic against HepG2 cells.Therefore, the decrease in cell viability after 48 hours incubation withPTX or PTX-loaded micelles can be attributed to PTX released from themicelles. The loaded micelle PTX-Polymer(8:8) (Example 13) killed HepG2cells more efficiently than the free PTX at most PTX concentrations. Asimilar trend was observed with loaded micelle PTX-Polymer(11:0)(Example 12) at low PTX concentrations. The killing efficiency of loadedmicelles PTX-Polymer(0:67) (Example 11), PTX-Polymer(11:30) (Example14), and PTX-Polymer(18:55) (Example 15) against HepG2 cells was greaterthan free PTX at all concentrations tested. These findings show that aPTX-loaded nanoparticulate formulation was more efficient than free PTXin killing HepG2 cells.

The above examples demonstrate that hydrophobiccholesterol-functionalized cyclic carbonate monomers, such as Chol-MTC,allow for efficient incorporation of cholesterol groups into acopolymers via metal-free organocatalytic ring opening polymerization,particularly when copolymerized with a diluent cyclic carbonyl monomersuch as TMC. Amphiphilic, biocompatible and/or biodegradable blockcopolymers can be formed using a mono-endcapped poly(alkylene glycol)initiators for the ring opening polymerization. The cholesterolfunctionalized block copolymers have low CMCs, and have high loadingcapacity for extremely hydrophobic and/or rigid drugs. The encapsulationof the drug can be accomplished via self-assembly without sonication orhomogenization using the above described dialysis techniques.Consequently, these polymeric materials are promising nanocarriers fordelivery of hydrophobic anticancer drugs such as PTX and other drugswith similar ring structures such as CYC and SPL.

ABA Triblock Copolymer Preparation

Several ABA triblock copolymers were prepared by a transition-metal freeorganocatalytic ring opening polymerization reaction using poly(ethyleneglycol) (PEG) as a macroinitiator and cholesteryl functionalized cycliccarbonate monomer (Chol-MTC) according to Scheme 3. Subscripts n′ and mof Scheme 3 are listed in Table 12 below.

In the notation TRI(p:m) of Scheme 3, p represents the two digits of thePEG name (e.g., p=20 for PEG20) used to form block B, and m representsthe average degree of polymerization of the cholesteryl repeat unit ofblock A.

The following procedure to prepare TRI(20:2.2) (Example 25 below) isrepresentative. In a 7 mL vial containing a magnetic stir bar in theglove box, Chol-MTC (135 mg, 219 micromoles, 8.6 equivalents), PEG20 (20kDa, 509 mg, 25.6 micromoles, 1.0 equivalent) and TU (8.0 mg, 21.6micromoles, 0.8 equivalents) were dissolved in dichloromethane (2.0 mL).To this solution, DBU (3.1 microliters, 3.2 mg, 20.8 micromoles, 0.8equivalents) was added to initiate polymerization. The reaction mixturewas allowed to stir at room temperature and aliquots of samples weretaken to monitor the monomer conversion and evolution of molecularweight by ¹H NMR spectroscopy and SEC. After 100 minutes, the reactionwas quenched by the addition of about 10 mg of benzoic acid and wasprecipitated into ice-cold diethyl ether (2×50 mL). The polymer wasdried in a tared vial for about 1 to 2 days, until a constant samplemass was obtained as white powder (470 mg, 73%).

Table 12 lists the properties of the ABA triblock copolymers. The weightpercent of the PEG chain in the ABA triblock copolymer, f_(PEG), wascalculated according to the equation:f _(PEG)={Mn^(PEG)/(Mn^(PEG)+[2×m×616])}×100%where Mn^(PEG) is the number average molecular weight of the PEGinitiator, and m is the average degree of polymerization of thecholesteryl repeat unit of block A.

TABLE 12 Triblock PEG PEG DP Chol DP f_(PEG) ^(d) Example Name Name(n′)^(b) (m)^(c) (%) 22 TRI(10:1.5) PEG10 247 1.5 84.8 23 TRI(10:1.9PEG10 247 1.9 81.0 24 TRI(20:0.8) PEG20 452 0.8 95.2 25 TRI(20:2.2)PEG20 452 2.2 87.9 26 TRI(35:2.4) PEG35 795 2.4 92.2 ^(a)number averagemolecular weight data from the supplier. ^(b)Average degree ofpolymerization of the ethylene oxide repeat unit of the B block(calculated). ^(c)Average degree of polymerization of the cholesterylrepeat unit of block A based on ¹H NMR spectroscopy. ^(d)Weight percentof PEG chain of the ABA triblock copolymer.

Thus, the A blocks of the ABA triblock copolymers in Table 12 had 0.8 to2.4 repeat units derived from Chol-MTC.

The PEG macroinitiators varied in weight average molecular weight of 10kDa to 35 kDa. The overall hydrophilicity of the polymers, as measuredby the weight percent of PEG, f_(PEG), of the ABA triblock copolymer,was about 85% to about 95%. In some instances, the resultant polymerscould be directly solubilized in aqueous solutions, if necessary withmild sonication/vortexing as an aid.

Critical Micelle Concentration (CMC) of ABA Triblock Copolymers

In some instances, the ABA triblock copolymers were able toself-assemble to form micelles in aqueous environment. For example, inDI water TRI(35:2.4) has a CMC of 16.6 mg/L (FIG. 7, graph). Forcomparison, diblock copolymer Polymer(11:30) has a CMC of 1.5 mg/L(FIG. 1. These values indicates the triblock copolymers can have astrong propensity for micelle formation, and that the micellar structureof the polymers can exist even at extensively dilute conditions, andpossibly the conditions of body fluids.

Preparation of FITC Loaded Micelles Example 27

Fluorescein isothiocyanate (FITC) was used as a model for incorporatinga biologically active substance in a composite hydrogel. 15 mg ofPolymer(11:30) and 3 mg of FITC were dissolved together in 2 mL DMF. Themixture was placed in a dialysis membrane tube with molecular weightcut-off (MWCO) of 1000 Da (Spectrum Laboratories, U.S.A.). The dialysisbag was then immersed in 1 L of de-ionized (DI) water at 4° C. for 2days. During the 2-day course, the dialysis medium was replaced after 3,6, and 24 hours. At the end of the dialysis process, the resultingmicelle solution was centrifuged at 4000 rpm for 5 minutes to removelarge aggregates. The particle size and zeta potential of the micelleswas then measured using a Zetasizer with dynamic light scatteringcapability (scattering angle: 90°) and equipped with a He—Ne laser beamat 658 nm (Malvern Instruments Zetasizer Nano ZS, UK).

FITC Loading Measurements

To determine the encapsulation efficiency of FITC in Polymer(11:30)micelles, the micelle suspension was mixed with DMSO (1:1) and thefluorescence intensity was measured at Ex/Em 485/528 nm using afluorescence spectrofluorometer (Horiba Scientific, Japan). Theencapsulation efficiency was calculated based on the ratio of the amountof FITC successfully encapsulated into the micelles to the amount ofFITC initially added during the micelle fabrication process. Table 13lists the properties of the FITC loaded micelle.

TABLE 13 Units of Units Initial Chol- of Loaded Loaded Loading LoadingLoaded FITC MTC TMC Micelle Micelle level efficiency Example Micelle(mg) (x) (y) Size (nm) PDI (wt %) (%) 27 FITC- 3 11 30 33 ± 0.4 0.08 ±0.01 19.5 59.2 Polymer(11:30)Formation of Composite Hydrogels

Hydrogels at known concentrations (4 wt % to 8 wt %) were prepared bydissolving the ABA triblock copolymers in either deionized water orloaded micelle solution at 25° C. for 4 hours. For polymers having ashorter PEG chain (10 kDa-20 kDa), ultrasonication was necessary toensure complete dissolution.

For the formation of composite hydrogel containing FITC, 1 ml of theloaded micelle suspension containing 0.36 mg/ml of FITC (Example 27) wasused to dissolve solid TRI(35:2.4) (40 mg). The resultant hydrogel wastransferred to dialysis membrane tubes with molecular weight cutoff(MWCO) of 1000 Da (Spectrum Laboratories, U.S.A.). The tubes wereimmersed in 20 ml of the release medium (i.e., PBS pH 7.4) and keptshaking on an orbital shaker at 100 rpm at 37° C. At various timeintervals, the release medium was removed and replaced with freshmedium.

Composite hydrogels were also prepared using CYC-loaded micellesprepared with Polymer(11:30) (Example 18). The concentration of thePolymer(11:30) and cyclosporine A in the micelle solution was 0.18% and0.036% respectively.

Table 14 lists the hydrogels and composite hydrogels formed.

TABLE 14 Total Triblock Solids Polymer Loaded Micelle Wt % Wt % Conc.Example (A) (B) A B (wt %) 28 TRI(20:2.2) 100 0 5 29 TRI(20:2.2) 100 0 830 TRI(35:2.4) FITC-Polymer (11:30) 95.9 4.1 4 31 TRI(35:2.4) 100 0 4 32TRI(35:2.4) CYC-Polymer (11:30) 95.9 4.1 4

Preparation of Free-FITC Loaded Hydrogel Example 33

FITC was loaded directly into triblock copolymer TRI(35:2.4) without thediblock copolymer using the following procedure. FITC and TRI(35:2.4)was mixed together in the presence of deionized water to form hydrogelwith FITC concentration of 0.36 mg/ml in 4 wt % hydrogel.

Rheological Experiments

The rheological analysis of the hydrogels was performed on an ARES-G2rheometer (TA Instruments, USA) equipped with a plate-plate geometry of8 mm diameter. Measurements were taken by equilibrating the gels at 25°C. and 37° C. between the plates at a gap of 1.0 mm. The data werecollected under controlled strain of 0.2 or 0.6% and a frequency scan of1.0 to 100 rad/s. Gelation properties of the polymer suspension wasmonitored by measuring the shear storage modulus (G′), as well as theloss modulus (G″), at each point. For shear-thinning studies, theviscosity of the hydrogels was monitored as function of shear rate from0.1 to 10 s⁻¹.

The influence of ABA copolymer concentration on storage modulus G′ isshown FIG. 8. An 8 wt % mixture of TRI(20:0.8) in deionized (DI) water(Example 29) has a storage modulus G′=2000 Pa, nearly 10-fold highercompared to a 5 wt % mixture of TRI(20:0.8) (G′=200 Pa) (Example 28).

A slight increase in the cholesteryl repeat units from 0.8 to 2.2drastically changes the solubility of the ABA triblock copolymers.TRI(20:2.2) has no visible solubility in DI water at 5 wt %, even afterovernight sonication. However, the insolubility can be reversed bydissolving the ABA triblock copolymer using the CYC loaded micellesuspension (Example 18) in place of DI water.

The 5 wt % composite hydrogel formed with TRI(20:2.2) (Example 30) had ahigher storage modulus at 25° C. (G′=2300 Pa) compared to a 5 wt %hydrogel of TRI(20:0.8) (Example 28) alone (G′=400 Pa) (FIG. 8).

Improvement in solubility of the ABA triblock copolymers was alsoobserved by increasing the PEG block from 20 kDa to 35 kDa. Forinstance, 4 wt % of TRI(35:2.4) (Example 31) was able to dissolve within4 hours when left standing at 25° C. without sonication. A 4 wt %solution of TRI(35:2.4) (Example 31) also formed hydrogel having ahigher storage modulus at 25° C. (G′=3500 Pa) (FIG. 9) compared to 5 wt% composite hydrogel of TRI(20:2.2) (Example 28) at 25° C. (G′=2300 Pa)(FIG. 8). A 4 wt % composite hydrogel of TRI(35:2.4) (Example 32) had astorage modulus G′ of about 3000 Pa at 25° C. (FIG. 9).

Dissolution using deionized water or cyclosporin A-loaded micellesuspension (Example 18) did not have a significant influence on thestorage and loss modulus of TRI(35:2.4).

Effects of temperature were also investigated. There were no changes tothe storage modulus (G′ 3500 Pa) and loss modulus (G″-150 Pa) ofTRI(35:2.4) (Example 31) when temperature was increased from 25° C.(FIG. 9) to 37° C. (FIG. 10).

FIG. 11 shows the reduction in viscosity with increasing shear rate atboth 25° C. and 37° C. of a hydrogel formed with 4 wt % TRI(35:2.4)(Example 31). The results indicate the shear-thinning characteristic ofthe gels, which results from the disruption of physical cross-linksbetween the polymer chains with the application of shear stress.

Scanning Electron Microscope (SEM) Imaging Of Hydrogel

To minimize morphological perturbations, the TRI(35:2.4) hydrogel(Example 31) was cryo-fixed by transferring the sample into a chamberfilled with liquid nitrogen. A 2-day freeze-drying process thenfollowed. The morphology of the gel was observed using the scanningelectron microscope (SEM) (Jeol JSM-7400F, Japan).

SEM imaging of the TRI(35:2.4) hydrogel (Example 31) formed in DI water(FIG. 12) shows the presence of long flexible fibers, most likely due tothe entanglement of the PEG chains. These long flexible fibers vary indiameter (˜0.1 to 1 micrometers) with anchors at the interaction sitesof cholesteryl units. In contrast, a composite hydrogel of TRI(35:2.4)(Example 32) prepared with CYC loaded Polymer(11:30) (Example 18) hasfibers that are shorter and thicker with larger diameters mostly around2 micrometers (FIG. 13).

In Vitro FITC Release

The release of FITC from the above-described composite hydrogel (Example30) was studied using the dialysis method. Prior to this, to analyze forthe amount of FITC released, the release medium was mixed with DMSO(1:1) and the fluorescence intensity was measured at E_(x)/E_(m) 485/528nm.

From fluorescence measurements, the encapsulation efficiency of FITC inPolymer(11:30) micelles was determined to be 59%. From the photograph ofFIG. 14, it can be seen that the composite hydrogel (Example 30)containing the FITC-loaded micelles was able to fluoresce (left vial,lighter shade in FIG. 14) under UV-illumination at 365 nm, while nofluorescence could be seen from a TRI(35:2.4) hydrogel (Example 33) thatcontained FITC that was not encapsulated in Polymer(11:30) micelles(right vial, darker shade in FIG. 14). This indicates a difference inthe solvent microenvironment for FITC, which results in changes to itsfluorescence characteristics.

The release of FITC from the composite hydrogel (Example 30) was studiedat 37° C. by immersing the gel in phosphate buffered saline (PBS). Forcomparison, FITC release from FITC loaded micelles (Example 27) and FITCrelease from the free-FITC loaded TRI(35:2.4) hydrogel (Example 33) werealso investigated. From the release profiles (FIG. 15), it can be seenthat the composite hydrogel (Example 30) provided greater sustainedrelease of FITC compared to FITC-loaded micelles (Example 27) alone andthe free-FITC in TRI(35:2.4) hydrogel (Example 33). About 60% of thedrug was released from the composite hydrogel in 17 hours while asimilar amount was released from the free-FITC/TRI(35:2.4) hydrogel in 2hours. Notably, the FITC loaded micelles (Example 27) show an extremelyhigh burst release with 60% of the drug escaping within the first 0.5hour.

CONCLUSIONS

Cholesteryl-containing biodegradable and biocompatible ABA-type triblockcopolymers that form hydrogels at relatively low polymer concentrationhave been developed. The viscoelastic properties of the hydrogels can betuned by varying the polymer concentration or composition, therebyproviding a storage modulus G′ having a value in a range of about 300 Pato about 3500 Pa. It has been demonstrated that composite hydrogelscomprising drug-loaded micelles and a hydrogel forming triblockcopolymer can provide sustained release of a hydrophobic drug. Thesetunable soft-composite gels serve as an attractive candidates for a widevariety of biomedical applications.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. When a range is used to express apossible value using two numerical limits X and Y (e.g., a concentrationof X ppm to Y ppm), unless otherwise stated the value can be X, Y, orany number between X and Y.

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiments were chosen and described in order to best explain theprinciples of the invention and their practical application, and toenable others of ordinary skill in the art to understand the invention.

What is claimed is:
 1. A composite hydrogel, comprising: an amphiphilictriblock copolymer (ABA), wherein i) the triblock copolymer comprisestwo independent peripheral hydrophobic A blocks and a hydrophiliccentral B block, ii) each of the A blocks comprises a steroidal repeatunit (repeat unit 1) comprising both a backbone carbonate and a sidechain comprising a steroid functional group, iii) each of the A blockshas a degree of polymerization of about 0.5 to about 4.0, and iv) the Bblock comprises a first poly(alkylene oxide) backbone; and a loadedmicelle comprising a biologically active substance and an amphiphilicdiblock copolymer (CD), wherein i) the diblock copolymer comprises ahydrophilic C block and a hydrophobic D block, ii) the C block comprisesa second poly(alkylene oxide) backbone, and iii) the D block comprises asteroidal repeat unit (repeat unit 2) comprising both a backbonecarbonate group and a side chain comprising a steroid functional group;wherein the amphiphilic triblock copolymer and the loaded micelle arebound by noncovalent interactions, and the composite hydrogel is capableof controlled release of a biologically active substance.
 2. Thecomposite hydrogel of claim 1, wherein at a temperature of 20° C. to 40°C. a 2 wt % to 5 wt % aqueous mixture of the composite hydrogel, basedon a total weight of the aqueous mixture, is a gel.
 3. The compositehydrogel of claim 1, wherein the triblock copolymer alone is not solublein water.
 4. The composite hydrogel of claim 1, wherein the triblockcopolymer alone does not form a hydrogel in water.
 5. The compositehydrogel of claim 1, wherein each of the A blocks has a degree ofpolymerization of about 0.8 to about 2.4.
 6. The composite hydrogel ofclaim 1, wherein each of the A blocks consists essentially of the repeatunit
 1. 7. The composite hydrogel of claim 1, wherein the steroidfunctional group of the repeat unit 1 is a cholesteryl group.
 8. Thecomposite hydrogel of claim 1, wherein the steroid functional group ofthe repeat unit 2 is a cholesteryl group.
 9. The composite hydrogel ofclaim 1, wherein the repeat unit 1 has a structure according to formula(7):

wherein t is an integer having a value of 0 to 6, t′ is an integerhaving a value of 0 to 6, t′ and t cannot both be zero, each Q¹ is anindependent monovalent radical selected from the group consisting ofhydrogen, halides, alkyl groups comprising 1 to 30 carbons, and arylgroups comprising 6 to 30 carbon atoms, and L′ is a divalent linkinggroup comprising one or more carbons.
 10. The composite hydrogel ofclaim 1, wherein the triblock copolymer comprises 5 wt % to 15 wt % ofthe repeat unit 1 based on a total weight of the triblock copolymer. 11.The composite hydrogel of claim 1, wherein the B block is a divalentpoly(ethylene oxide) chain having a number average molecular weight (Mn)of about 10000 to about
 35000. 12. The composite hydrogel of claim 1,wherein the C block is a monovalent endcapped poly(ethylene oxide)having a number average molecular weight of about
 5000. 13. Thecomposite hydrogel of claim 1, wherein the triblock copolymer has acritical micelle concentration (CMC) in deionized water between 0 mg/Land 20 mg/L.
 14. The composite hydrogel of claim 1, wherein the diblockcopolymer has a critical micelle concentration (CMC) in deionized waterbetween 0 mg/L and 5 mg/L.
 15. The composite hydrogel of claim 1,wherein the biologically active substance is a hydrophobic drug.
 16. Thecomposite hydrogel of claim 15, wherein the drug is cyclosporin A (CYC).17. A method, comprising: mixing an amphiphilic triblock copolymer (ABA)with a loaded micelle, the loaded micelle comprising i) a biologicallyactive substance and ii) an amphiphilic diblock copolymer (CD), therebyforming a composite hydrogel capable of controlled release of thebiologically active substance; wherein the triblock copolymer (ABA)comprises two independent peripheral hydrophobic A blocks and ahydrophilic central B block, wherein i) each of the A blocks comprises asteroidal repeat unit (repeat unit 1) comprising both a backbonecarbonate and a side chain comprising a steroid functional group, ii)each of the A blocks has a degree of polymerization of about 0.5 toabout 4.0, and iii) the B block comprises a first poly(alkylene oxide)backbone, the diblock copolymer (CD) comprises a hydrophilic C block anda hydrophobic D block, wherein i) the C block comprises a secondpoly(alkylene oxide) backbone, and ii) the D block comprises a steroidalrepeat unit (repeat unit 2) comprising both a backbone carbonate and aside chain steroid functional group, and the triblock copolymer and theloaded micelle are bound by noncovalent interactions.
 18. The method ofclaim 17, wherein the triblock copolymer and the diblock copolymer aremixed in water.
 19. The method of claim 18, wherein the triblockcopolymer alone is not soluble in the water until mixed with the loadedmicelle.
 20. The method of claim 17, wherein the triblock copolymer isformed by an organocatalyzed ring opening polymerization of a cycliccarbonate monomer initiated by poly(alkylene glycol), the cycliccarbonate monomer having the formula (6):

wherein t is an integer having a value of 0 to 6, t′ is an integerhaving a value of 0 to 6, t′ and t cannot both be zero, each Q¹ is anindependent monovalent radical selected from the group consisting ofhydrogen, halides, alkyl groups comprising 1 to 30 carbons, and arylgroups comprising 6 to 30 carbon atoms, and L′ is a divalent linkinggroup comprising one or more carbons.
 21. A composite hydrogel forcontrolled release of a biologically active substance, comprising: anamphiphilic triblock copolymer (ABA) having the structure:

wherein each m is an independent number having a value of about 0.8 toabout 2.5, and n′ has a value of about 200 to about 800; and a loadedmicelle comprising i) the biologically active substance and ii) anamphiphilic diblock copolymer (CD) having the structure:

wherein n has a value of about 100 to about 120, x has a value of about11, and y has a value of about 30, and wherein the amphiphilic triblockcopolymer and the loaded micelle are bound by noncovalent interactions.22. The composite hydrogel of claim 21, wherein the biologically activesubstance is a drug.
 23. The composite hydrogel of claim 22, wherein thedrug is selected from the group consisting of cyclosporin A, paclitaxel,spironolactone, and combinations thereof.
 24. A triblock copolymerhaving the formula (19)

wherein each m is an independent number having a value of about 0.8 toabout 4, n′ has a value of about 200 to about 800, each t is anindependent integer having a value of 0 to 6, each t′ is an independentinteger having a value of 0 to 6, t′ and t cannot both be zero in anyrepeat unit, each Q¹ is an independent monovalent radical selected fromthe group consisting of hydrogen, halides, alkyl groups comprising 1 to30 carbons, and aryl groups comprising 6 to 30 carbon atoms, each L′ isan independent linking group selected from the group consisting of asingle bond and divalent radicals comprising 1 to 30 carbons, and eachS′ is an independent steroid group.
 25. The triblock copolymer of claim24, wherein S′ is cholesteryl.
 26. The triblock copolymer of claim 24,wherein the triblock copolymer has the formula (20),

wherein each m is an independent number having a value of about 0.8 toabout 4, n′ has a value of about 200 to about 800, each t is anindependent integer having a value of 0 to 6, each t′ is an independentinteger having a value of 0 to 6, t′ and t cannot both be zero in anyrepeat unit, each Q¹ is an independent monovalent radical selected fromthe group consisting of hydrogen, halides, alkyl groups comprising 1 to30 carbons, and aryl groups comprising 6 to 30 carbon atoms, each L″ isan independent linking group selected from the group consisting of asingle bond and divalent radicals comprising 1 to 30 carbons, and eachS′ is an independent steroid group.
 27. The triblock copolymer of claim24, wherein the triblock copolymer has the formula (21),

wherein each m is an independent number having a value of about 0.8 toabout 4.0, and n′ has a value of about 200 to about 800.